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WO2003014375A2 - Molecules de sonde d'acide nucleique et methodes d'utilisation de ces dernieres - Google Patents

Molecules de sonde d'acide nucleique et methodes d'utilisation de ces dernieres Download PDF

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Publication number
WO2003014375A2
WO2003014375A2 PCT/US2002/025319 US0225319W WO03014375A2 WO 2003014375 A2 WO2003014375 A2 WO 2003014375A2 US 0225319 W US0225319 W US 0225319W WO 03014375 A2 WO03014375 A2 WO 03014375A2
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nucleic acid
acid sensor
target
sensor molecule
molecule
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WO2003014375A3 (fr
Inventor
Martin Stanton
David Epstein
Nobuko Hamaguchi
Markus Kurz
Tony Keefe
Charles Wilson
Dilara Grate
Kristin A. Marshall
Thomas Mccauley
Jeffrey Kurz
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Archemix Corp
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Archemix Corp
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Priority claimed from US09/952,680 external-priority patent/US20030087239A1/en
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Publication of WO2003014375A2 publication Critical patent/WO2003014375A2/fr
Publication of WO2003014375A3 publication Critical patent/WO2003014375A3/fr
Anticipated expiration legal-status Critical
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    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
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    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates generally to nucleic acids and more particularly to nucleic acid sensor molecules containing a catalytically active domain that is modulated upon recognition of a target by a target modulation domain of the nucleic acid sensor molecule.
  • nucleic acids can adopt complex three- dimensional structures. These three-dimensional structures are capable of specific recognition of target molecules and, furthermore, of catalyzing chemical reactions. Nucleic acids will thus provide candidate detection molecules for diverse target molecules, including those which that do not naturally recognize or bind to DNA or RNA.
  • a nucleic acid which binds to a non-nucleic acid target molecule through non- Watson-Crick base pairing is termed an aptamer.
  • aptamer selection combinatorial libraries of oligonucleotides are screened in vitro to identify oligonucleotides, or aptamers, which bind with high affinity to pre-selected targets.
  • small biomolecules e.g., amino acids, nucleotides, NAD, S-adenosyl methionine, chloramphenicol
  • large biomolecules thrombin, Ku, DNA polymerases
  • Aptamer biosensors have been used to detect specific analyte molecules.
  • fluorescently labeled anti-thrombin aptamers attached to a glass surface have been used to directly detect the presence of thrombin proteins in a sample by detecting changes in the optical properties of the aptamers (Potyrailo, et al., 1998).
  • continuous binding of thrombin to the labeled aptamer is requisite for detection to occur, since the concentration of thrombin in a test sample is monitored by directly detecting fluorescent emission of the aptamer-ligand complex.
  • a limitation with this type of aptamer-derived biosensor is that ligand-mediated changes in secondary structure were engineered into the aptamer molecule via a laborious engineering process in which four to six nucleotides were added to the 5' end of the aptamer that was complementary to the bases at the 3' end of the thrombin binding region. In the absence of thrombin, this structure forms a stem loop structure, while it forms a G-quartet structure in the presence of thrombin. Fluorescent and quenching groups attached to the 5' and 3' end signal this change.
  • assay sensitivity and hence the limit of detection is set empirically by the affinity of the aptamer- ligand complex, the KD value.
  • affinity of the aptamer- ligand complex the KD value.
  • antibody-based detection requires continuous ligand binding and ligand-antibody complex formation for the generation of a detectable signal.
  • antibody methods such as ELISA or competitive RIA, while robust, are restricted in utility because these methods require that heterogeneous assay conditions be employed: 1] detection is done on a solid surface; 2] in most applications both a capture antibody and detection antibody are required; 3] for ELISA-based protein detection methods, the antibodies must recognize the folded, native structure of the protein that is present in cell or tissue isolates and; 4] antibody and protein based detection methods have not been described for intracellular or in vivo based analyte detection.
  • These assays include 1] the initial discovery of a drug target through protein or metabolite profiling, 2] the subsequent use of that same drug target in the discovery of drug leads through high throughput screening and, 3] the optimization of drug leads against that same drug target through an evaluation of lead efficacy in mechanistic cellular and in vivo animal assays.
  • nucleic acid molecular sensors that can function in environments and formats that includes but are not limited to solution-based detection (homogeneous in vitro biochemical assays or in vivo cellular and animal assays), chip-based (heterogeneous in vitro assays on solid surfaces), and assays in complex biological isolates from blood plasma, cell lysates or tissue extracts.
  • Nucleic acid-based detection schemes have exploited the ligand-sensitive catalytic properties of some nucleic acids, e.g., such as ribozymes.
  • Ribozyme-based prototype nucleic acid sensor molecules have been designed both by engineering and by in vitro selection methods. Engineering methods exploit the apparently modular nature of RNA structures; these sensors couple molecular recognition to signaling by simply joining individual target-modulation and catalytic RNA domains through a double-stranded or partially double-stranded RNA linker.
  • ATP sensors for example, were created by appending the previously-selected, ATP-binding aptamer-derived sequences (Sassanfar and Szostak, 1993) to either the self-cleaving hammerhead ribozyme (Tang and Breaker, 1997) or the LI self-ligating ribozyme (Robertson and Ellington 2000). Robertson and Ellington (2000) have demonstrated that the enzymatic activity of a ligase ribozyme (derived from the LI ligase described in Robertson and Ellington (1999)) can be modulated by a small molecule ligand, or small molecule target recognition.
  • the ligase ribozyme can be employed as a nucleic acid sensor molecule and used to detect the presence and level of its cognate ligand by monitoring the ligation of a small, labeled second oligonucleotide substrate on to the ribozyme.
  • a distinct feature of this detection method is that the actual detection event, e.g., monitoring oligonucleotide substrate ligation to the ribozyme, occurs after the ligand interacts with the nucleic sensor molecule.
  • the ribozyme-based ligand detection method of Robertson and Ellington does not require continuous binding of the ligand to the sensor molecule in order to generate a detectable signal.
  • radiolabeled hammerhead ribozymes which undergo cleavage upon binding to a ligand, have been used to detect ligand by monitoring the release of the label from the ribozyme (Soukup, et al., 2000, and Breaker, 1998).
  • Limitations of the use of ligand modulated hammerhead ribozymes described by Soukup, et al, 2000, and Breaker, 1998 include: 1] the need for a two-step detection method for determining the enzymatic activity of the surface- immobilized hammerhead-derived sensors; 2] the need for radiometric determination of hammerhead activity in both solution and solid-surface based assay formats; 3] the need for significant chemical and structural modification of the hammerhead-based biosensor to render them suitable for optical based detection methods.
  • Ellington and Roberts (1999) describe a region of the LI ligase that is required for allosteric ribozyme function, termed the effector oligonucleotide binding domain. It was postulated that the effector oligonucleotide binding domain of the ligase formed complementary base pairing interactions with the oligonucleotide substrate binding site, driving the ribozyme into an inactive conformation.
  • the effector oligonucleotide when added to the LI ligase activates (fotct) the enzyme by over 10,000 fold over the LI ligase reaction measure in the absence of effector ( ⁇ unact).
  • the native LI -ligase has a switch factor (k cx/k amct ) greater than 10,000, which determines the sensitivity of a ribozyme-based detection method.
  • the ligase activity of the deletion mutant is only 3-5 fold lower than the ligase activity of LI ligase with the effector oligonucleotide bound to the effector oligonucleotide binding domain (Ellington and Robertson (1999). This indicates that LI ligases deleted of the effector oligonucleotide binding domain may not be not subject to further allosteric regulation.
  • a hindrance to the development of LI ligase-based biosensor technology is the lack of a general method for the generation of biosensors that can work in multiple assay and detection formats required of solution-based and chip-based biosensors and, those that can work in multiplexed formats and in complex biological extracts.
  • the nucleic acid sensor molecules of the present invention are used to monitor the presence or concentration of various target molecules (proteins, post-translationally modified forms of proteins, peptides, nucleic acids, oligosaccharides, nucleotides, metabolites, drugs, toxins, biohazards, and ions) and function in solution-based homogeneous assays using optical or other detection methods; in solution-based homogeneous assays using PCR-based or other nucleotide amplification-based detection methods; in homogeneous intracellular assays using PCR-based detection or other nucleotide amplification-based detection methods; in heterogeneous assays (surface- immobilized nucleic acid sensor molecules and surface-capture nucleic acid sensor molecules) using optical detection methods; and in heterogeneous assays (surface- immobilized nucleic acid sensor molecules and surface-capture nucleic acid sensor molecules) using PCR-based detection or other nucleotide amplification-based detection methods.
  • nucleic acid sensor molecules of the present invention were developed through a combination of engineering and selection methods that are now shown to be useful for identifying nucleic acid sensor molecules against a wide variety of target molecules including proteins (including specific post-translationally modified forms of proteins) peptides, nucleic acids, oligosaccharides, nucleotides, metabolites, drugs, toxins, biohazards, ions, carbohydrates, glycoproteins, hormones, receptors, antibodies, viruses, transition state analogs, cofactors, dyes growth factory nutrients, etc.
  • proteins including specific post-translationally modified forms of proteins
  • nucleic acid sensor molecules are based on cis-cleaving hammerhead ribozymes that have been designed to work as optical signaling molecules in a homogeneous assay format, and utilize fluorescence and FRET based methods of signal generation and detection.
  • nucleic acid sensor molecules are based on cis- cleaving hammerhead ribozymes that have been designed to work as optical signaling molecules affixed to a solid-support, and utilize fluorescence and FRET based methods of signal generation and detection.
  • the nucleic acid sensor molecules are based on cis-cleaving hammerhead ribozymes that have been designed to work as optical signaling molecules affixed to a solid-support and utilize surface plasmon resonance methods of signal generation and detection. In one embodiment of the invention the nucleic acid sensor molecules are based on a
  • 3-piece LI ligase ribozyme that retains the effector oligonucleotide binding domain, and has been designed to detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule, in solution using quantitative PCR-based methods.
  • the nucleic acid sensor molecules are based on a 2-piece LI ligase ribozyme that couples the effector oligonucleotide substrate to the oligonucleotide substrate forming an oligonucleotide supersubstrate and, has been designed to detect target molecules, as a function of oligonucleotide supersubstrate ligation to the nucleic acid sensor molecule, in solution using quantitative PCR-based methods.
  • the nucleic acid sensor molecules are based on a 1-piece LI ligase ribozyme that deletes the effector oligonucleotide binding domain.
  • the 1- piece ligase is designed to self-ligate or circularize by joining the 3'-OH end and the PPP- 5'-end of the ligase and detects target molecules, as a function of circularization of the nucleic acid sensor molecule, in solution using PCR-based methods.
  • the nucleic acid sensor molecules are based on a 3-piece LI ligase ribozyme that retains the effector oligonucleotide binding domain, and has been designed to detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule immobilized on a solid support and where the detection uses fluorescence-based methods.
  • the 3-piece LI ligase nucleic acid sensor molecules detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule in solution and then are subsequently captured on a solid support and detected using fluorescence-based methods.
  • the 3-piece LI ligase nucleic acid sensor molecules detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule in solution and then are subsequently captured on a solid support and detected using radiometric-based methods.
  • the nucleic acid sensor molecules are based on a 2-piece LI ligase ribozyme and has been designed to detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule immobilized on a solid support and where the detection uses optical methods such as fluorescence-based methods.
  • the 2-piece LI ligase nucleic acid sensor molecules detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule in solution and then are subsequently captured on a solid support and detected directly using fluoresence-based methods.
  • the 2-piece LI ligase nucleic acid sensor molecules detect target molecules, as a function of oligonucleotide substrate ligation to the nucleic acid sensor molecule in solution and then are subsequently captured on a solid support and directly detected using radiometric-based methods.
  • the nucleic acid sensor molecules is based on a 1-piece LI ligase ribozyme that is transfected into a mammalian cell.
  • the 1-piece ligase is designed to self-ligate or circularize by joining the 3'-OH end and the PPP-5'-end of the ligase and detects intracellular target molecules, as a function of circularization of the nucleic acid sensor molecule.
  • the circularized ligase molecules are then re-isolated from the cellular lysate and the amount of target present in the cell is quantified using solution based nucleotide amplification methods.
  • the invention includes a nucleic acid sensor molecule which includes a target modulation domain which recognizes a target molecule, a linker domain, a catalytic domain, and an optical signal generating unit.
  • the nucleic acid sensor molecule of the invention has an optical signal generating unit that includes at least one signaling moiety. In another embodiment, the nucleic acid sensor molecule of the invention has an optical signal generating unit which includes at least a first signaling moiety and a second signaling moiety. In another embodiment, the first and second signaling moieties change proximity to each other upon recognition of a target by the target modulation domain. In another embodiment, the first and second signaling moieties include a fluorescent donor and a fluorescent quencher, and recognition of a target by the target modulation domain results in an increase in detectable fluorescence of said fluorescent donor.
  • the first signaling moiety and said second signaling moiety include fluorescent energy transfer (FRET) donor and acceptor groups, and recognition of a target by the target modulation domain results in a change in distance between said donor and acceptor groups, thereby changing optical properties of said molecule.
  • the invention includes a nucleic acid sensor molecule where the optical signal generating unit consists essentially of a first signaling moiety which changes conformation upon recognition of a target by the target modulation domain, thereby resulting in a detectable optical signal.
  • the nucleic acid sensor molecule includes at least one modified nucleic acid.
  • the catalytic domain of the nucleic acid sensor molecule includes an endonucleolytic ribozyme.
  • the endonucleolytic ribozyme can be, for example, a cw-endonucleolytic ribozyme or a tr ⁇ '-endonucleolytic ribozyme.
  • the endonucleolytic ribozyme is a hammerhead ribozyme.
  • the catalytic domain of the nucleic acid sensor includes a self-ligating ribozyme.
  • the elf-ligating ribozyme can be, for example, a cz ' s-ligase ribozyme or a trar ⁇ -ligase ribozyme.
  • the self-ligating ribozyme can be, e.g., a 1-piece ligase, 2-piece ligase or 3-piece ligase.
  • the target modulation domain recognizes a target that is selected from proteins, post-translationally modified forms of proteins, peptides, nucleic acids, oligosaccharides, nucleotides, metabolites, drugs, toxins, biohazards, ions, carbohydrates, polysaccharides, hormones, receptors, antigens, antibodies, viruses, metabolites, co-factors, drugs, dyes, nutrients, or growth factors.
  • the target modulation domain recognizes a protein or a post- translationally modified protein.
  • the target modulation domain recognizes a post- translationally modified protein, wherein the post-translational modification can be a phosphorylation, prenylation, glycosylation, methionine removal, N-acetylation, acylation, acylation of cysteines, myristoylation, alkylation, ubiquitinylation, prolyl-4-hydroxylation, carboxylation of glutaminyl residues, advanced glycoslylation, deamination of glutamine and asparagine, addition of glycophosphatidylinositol, disulfide bond formation, hydroxylation, or lipidation.
  • the post-translational modification can be a phosphorylation, prenylation, glycosylation, methionine removal, N-acetylation, acylation, acylation of cysteines, myristoylation, alkylation, ubiquitinylation, prolyl-4-hydroxylation, carboxylation of glutaminyl residues, advanced glycoslylation, deamination of glutamine
  • the target is a protein kinase. In other embodiments, the target is a phosphorylated protein kinase.
  • the phosphorylated protein kinase can be a monophosphorylated protein kinase or a diphosphorylated protein kinase.
  • the target protein is ERK, such as ERK1 or ERK2.
  • the post-translationally modified protein is ppERK, such as such as ppERKl or ppERK2..
  • the nucleic acid sensor molecule includes a target modulation domain which recognizes a component of a MAP kinase pathway, a product of a MAP kinase pathway, a MAP kinase pathway associated protein, or an extracellular component of a MAP kinase pathway.
  • the target modulation domain recognizes a component of the ERKl/2 MAP kinase pathway, the JNK MAP kinase pathway, or the P38 MAP kinase pathway.
  • the target modulation domain recognizes an endogenous form of a MAP Kinase (MEK), an endogenous form of a MAP Kinase Kinase (MEKK), or an endogenous form of MAP Kinase Kinase Kinase, (MEKKK).
  • the target modulation domain recognizes an endogenous form of RAF kinase.
  • the target modulation domain recognizes a Ras protein, a phosphatase, s a GTP binding protein, a GPCR, a cytokine, a growth factor, a cellular metabolite, or a small molecule.
  • the nucleic acid sensor molecule includes RNA, DNA, or both RNA and DNA.
  • the invention also relates to compositions containing a nucleic acid sensor molecule of the invention and a buffer.
  • the invention includes a composition containing a nucleic acid sensor molecule and a tissue extract, a cell extract or an in vitro cell culture.
  • a composition of the invention also includes an RNase inhibitor, such as, for example, Va-riboside, vanadyl, tRNA, polyU, RNaseln or RNaseOut.
  • a composition of the invention is substantially RNase-free.
  • the invention also relates to a composition which includes at least one nucleic acid sensor molecule affixed to a substrate.
  • the substrate is glass, gold or other metal, silicon or other semiconductor material, nitrocellulose, nylon, or plastic.
  • the nucleic acid sensor molecule is covalently attached to the substrate.
  • the nucleic acid sensor molecule is non-covalently attached to the substrate.
  • the nucleic acid sensor molecule is immobilized to the substrate via hybridization of a terminal portion of the nucleic acid sensor molecule to an oligonucleotide that is bound to the surface of the substrate.
  • a composition includes a plurality of nucleic acid sensor molecules immobilized to the substrate via hybridization of a terminal portion of the nucleic acid sensor molecule to an array of oligonucleotides bound to the substrate at spatially discrete regions. In some embodiments, at least two members of this plurality each recognize different target molecules.
  • the substrate can include, for example, at least 50 nucleic acid sensor molecules. In other embodiments, the substrate includes at least 250 nucleic acid sensor molecules, at least 500 nucleic acid sensor molecules, or at least 5000 nucleic acid sensor molecules.
  • the invention includes a system for detecting a target molecule which includes a composition according to the invention and a detector in optical communication with the composition, where the detector detects changes in the optical properties of the composition.
  • the system further includes a light source in optical communication with the composition.
  • the system also includes a processor for processing optical signals detected by the detector.
  • the system for detecting a target molecule includes a plurality of nucleic acid sensor molecules where at least two of the biosensor molecules each recognize different target molecules.
  • the invention includes a method of identifying or detecting a target molecule in a sample by contacting a sample suspected of containing a target molecule with a nucleic acid sensor molecule of the invention, wherein a change in the signal generated by the optical signal generating unit indicates the presence of target in the sample.
  • the method further includes quantifying the change signal generated by the optical signal generating unit to quantify the amount of target molecule in the sample.
  • the sample is an environmental sample, biohazard materials, organic samples, drugs and toxins, flavors and fragrances, and biological samples.
  • the sample is a biological sample such as cells, cell extracts or lysates, tissues or tissue extracts, bodily fluids, serum, blood and blood products.
  • the method of identifying or detecting a target molecule in a sample detects of identifies proteins, post-translationally modified forms of proteins, peptides, nucleic acids, oligosaccharides, nucleotides, metabolites, drugs, toxins, biohazards, ions, carbohydrates, polysaccharides, hormones, receptors, antigens, antibodies, viruses, metabolites, co-factors, drugs, dyes, nutrients, or growth factors.
  • the method of the invention detects of identifies proteins or post- translationally modified forms of proteins.
  • the target is a post- translationally modified protein, where the post-translation modifications is phosphorylation, prenylation, glycosylation, methionine removal, N-acetylation, acylation, acylation of cysteines, myristoylation, alkylation, ubiquitinylation, prolyl-4-hydroxylation, carboxylation of glutaminyl residues, advanced glycoslylation, deamination of glutamine and asparagine, addition of glycophosphatidylinositol, disulfide bond formation, hydroxylation, or lipidation.
  • the post-translation modifications is phosphorylation, prenylation, glycosylation, methionine removal, N-acetylation, acylation, acylation of cysteines, myristoylation, alkylation, ubiquitinylation, prolyl-4-hydroxylation, carboxylation of glutaminyl residues, advanced glycoslylation, deamination of glutamine and asparagine, addition of glycophosphatidy
  • the target is a protein kinase.
  • the target is a phosphorylated protein kinase.
  • the invention includes a diagnostic system for identifying or detecting a target molecule, where the diagnostic system includes a nucleic acid sensor molecule of the invention and a detector in communication with the nucleic acid sensor molecule, wherein the detector detects changes in the signal generated by the optical signal generating unit of the nucleic acid sensor.
  • the diagnostic system further includes a processor for processing signals detected by the detector.
  • the invention includes a method of identifying or detecting a protein kinase in a sample by contacting a sample suspected of containing a protein kinase with a nucleic acid sensor molecule, wherein said nucleic acid sensor molecule has a target recognition domain that recognizes a protein kinase, and wherein a change in the signal generated by the optical signal generating unit indicates the presence of protein kinase in the sample.
  • the method further includes quantifying the amount of signal generated by the optical signal generating unit to quantify the amount of protein kinase in the sample.
  • the invention provides a method of identifying a modulator of protein kinase activity by contacting a test agent with a protein kinase and nucleic acid sensor molecule, wherein the nucleic acid sensor molecule has a target recognition domain that recognizes a protein kinase, recognition of the protein kinase by the target recognition domain of the nucleic acid sensor molecule results in a change in the signal generated by the optical signal generating unit and wherein changes in the signal generated by the optical signal generating unit in the presence and absence of the test agent indicates the test agent is a modulator of the protein kinase activity.
  • the catalytic domain of the nucleic acid sensor molecule includes a cz ' s-ligase ribozyme or a tr ⁇ r ⁇ -ligase ribozyme.
  • the invention provides a nucleic acid sensor molecule which includes a target modulation domain that recognizes ERK, a catalytic domain that includes a ligase or cis-hammerhead, and a linker domain that links the target modulation domain and the catalytic domain.
  • the invention includes a nucleic acid sensor molecule which includes a target modulation domain that recognizes phosphoERK, a catalytic domain that includes a ligase or a cis-hammerhead, and a linker domain that links the target modulation domain and the catalytic domain.
  • the invention provides a nucleic acid sensor molecule which includes a target modulation domain that recognizes lysozyme, a catalytic domain that includes a 1-piece cis-ligase and a linker domain that links the target modulation domain and the catalytic domain.
  • the invention provides a nucleic acid sensor molecule which includes a target modulation domain that recognizes any one of cCMP, cAMP, or cGMP, a catalytic domain, and a linker domain that links the target modulation domain and the catalytic domain, wherein the nucleic acid sensor molecule includes an optical signal generating unit or a non-radioactive detectable label.
  • the nucleic acid sensor molecule includes an optical signal generating unit.
  • the nucleic acid sensor molecule includes a detectable label.
  • the label is a radioactive label, such as, for example, 32 P, 33 P, 14 C, 35 S, 3 H, or 125 I.
  • the nucleic acid sensor molecule comprises a fluorescent label, such as, for example, fluorescein, DABCYL, or a green fluorescent protein (GFP) moiety.
  • the optical signal generating unit includes a fluorescent moiety and a quenching moiety, wherein recognition by the target modulation domain causes causes a change in detectable fluorescence by the optical signal generating unit.
  • the nucleic acid sensor molecule includes an enzymatic label, or an affinity capture tag label.
  • the nucleic acid sensor molecule includes a target modulation domain recognizes ERK1, ERK2 or both.
  • the nucleic acid sensor molecule includes a target modulation domain and catalytic domain are as shown by SEQ ID NO. 80, and the linker is randomized.
  • the nucleic acid sensor molecule includes a target modulation domain and a catalytic domain as shown in any one of SEQ ID NOS. 47, 118 and 119, and the linker is randomized.
  • the invention provides a nucleic acid sensor molecule that recognizes ERK having the SEQ ID NO. 90-95, 108-1 16, 131-133, 140-295, 349, 351, or 356.
  • the invention provides a nucleic acid sensor molecule that recognizes phospoERK having the SEQ ID NO. 5-8, 37-39, 44-45, 81-89, 96-100, 121-130, 352, or 353.
  • the invention provides a nucleic acid sensor molecule that recognizes any one of cCMP, cAMP or cGMP, having the SEQ ID NO. 40-43, 103, or 135- 139.
  • the invention provides a nucleic acid sensor molecule that recognizes lysozyme, having the SEQ ID NO. 46, 47, 76, or 105-107.
  • the invention provides a 1-piece ligase ribozyme including a target modulation domain that recognizes a target, a linker domain, and a catalytic domain wherein the 5' and 3' ends of the ligase ligate to each other upon recognition of the target by the modulation domain.
  • the invention provides a 2-piece ligase ribozyme including a target modulation domain that recognizes a target, a linker domain, and a catalytic domain including an oligonucleotide substrate ligation site and and oligonucleotide supersubstrate binding domain, wherein upon recognition of the target by the modulation domain, the 3' end of the an oligonucleotide supersubstrate is ligated to the 5' end of the oligonucleotide substrate ligation site.
  • the invention provides a 3-piece ligase ribozyme including a target modulation domain that recognizes a target, a linker domain, and a catalytic domain comprising comprising an oligonucleotide substrate binding domain capable of binding an oligonucleotide substrate and an effector-oligonucleotide binding site capable of binding an effector oligonucleotide, wherein upon recognition of the target by the modulation domain, and in the presence of binding of the effector oligonucleotide to the effector-oligonucleotide binding site, then the 3' end of the oligonucleotide substrate is ligated to the 5' end of the ligase.
  • the invention also provides a 1-piece ligase ribozyme comprising the nucleic acid sensor molecule shown in any one of SEQ ID NOS. 47, 105-107, 119.
  • the invention also provides a 2-piece ligase ribozyme comprising the nucleic acid sensor molecule shown in any one of SEQ ID NOS. 347, 349, and 351.
  • the invention further provides a 3-piece ligase ribozyme comprising the nucleic acid sensor molecule shown in any one of SEQ ID NOS. 46, 75, 76, 108-116, 118, 121-130, and 352.
  • Figure 1 depicts methodology for selecting nucleic acid sensor molecules of the invention.
  • Figure IA is a flow diagram showing a method for selecting nucleic acid sensor precursor molecules having a target molecule activatable ligase activity according to one embodiment.
  • Figure IB is a flow diagram showing a gel-based method for selecting nucleic acid sensor precursor molecules having a target molecule activatable endonuclease activity according to one embodiment.
  • Figures 2A and B show a nucleic acid sensor molecule (SEQ ID NO:46) according to one embodiment, in which the catalytic site includes a ligase site.
  • Figure 2A shows the conformation of the target molecule (SEQ ID NO:46) bound form of the nucleic acid sensor molecule with an effector oligo hybridized to its 3' end (SEQ ID NO:51).
  • Figure 2B shows the conformation of the non-target bound form of the nucleic acid (SEQ ID NO:46) sensor molecule.
  • Figures 3A and B show a nucleic acid sensor molecule (SEQ ID NO:47) derived from the nucleic acid sensor precursor molecule shown in Figures 2 A and B in which first and second nucleotides are labeled with first and second signaling moieties (F and D, respectively).
  • Figure 4 is a flow diagram showing a method for selecting nucleic acid sensor molecules having a target molecule activatable self-cleavage activity according to one embodiment.
  • Figures 5A and B show a nucleic acid sensor molecule according to one embodiment, in which the catalytic site includes a self-cleavage site which is the catalytic core of a hammerhead ribozyme.
  • Figure 5 A shows the conformation of the target molecule bound form of the nucleic acid sensor molecule (SEQ ID NO:48).
  • Figure 5B shows the conformation of the non-target bound form of the nucleic acid sensor molecule (SEQ ID NO:48).
  • Figures 6A and B show a nucleic acid sensor molecule derived from the nucleic acid sensor molecule shown in Figures 3A and B in which first and second nucleotides are labeled with first and second signaling moieties (F and D, respectively) (SEQ ID NO:49).
  • Figure 7 is a schematic diagram illustrating pathway target molecules according to one embodiment.
  • Figure 8 is a flow chart showing steps in a drug optimization method according to one embodiment, in which nucleic acid sensor molecules are used at each step in the method.
  • Figure 9A shows an example of a self-cleaving nucleic acid sensor bound to a solid support when used in an epi-illuminated FRET detection scheme.
  • Figure 9B shows the same sensor in an epi-illuminated beacon configuration, with the acceptor fluorophore replaced by a quencher group.
  • Figure 9C shows the same sensor in an TIR-illuminated beacon configuration.
  • Figure 10A shows an example of a self-ligating nucleic acid sensor bound to a solid support when used in a TIR-illuminated detection scheme where there is a signal increase upon target binding.
  • Figure 1 OB shows the same sensor in an epi-illuminated configuration, where target binding is detected by monitoring changes of the fluorophore bound to the substrate at the surface of the array.
  • Figure IOC shows the same epi-illuminated configuration, where target binding is detected by monitoring changes in the fluorescence polarization.
  • Figure 11 illustrates the use of beads in a homogeneous assay format utilizing a self- ligating nucleic acid sensor.
  • Figure 11 A shows the beads prior to target binding and ligation (no emission from acceptor).
  • Figure 1 IB shows the beads after target binding and ligation (emission from acceptor detected).
  • Figure 12 shows a schematic of a previously constructed scanning detection system utilizing TIR laser evanescent wave excitation in either large area illumination/CCD imaging mode, or scanned spot/PMT imaging mode. The schematic shows how an array can be scanned and FP or fluorescence intensity data extracted.
  • Figure 13 shows a schematic of fluorescence data generated by a biosensor array using the indicated nucleic acid sensors and target molecules.
  • Figures 14 shows a schematic showing different catalytic platforms for detection methods for nucleic acid sensor molecules.
  • Figure 15 is a schematic showing a ligase nucleic acid sensor molecule sensor system. It shows SEQ ID NO:76 across the top, and SEQ ID NO:77 hybridized to a portion of SEQ ID NO:76 and bound to an insulating moiety.
  • Figure 16 is a schematic showing a hammerhead (endonuclease) Nucleic acid sensor molecule sensor system. It has a nucleic acid sensor molecule sensor (SEQ ID NO:78), and SEQ ID NO:79 hybridizing to a portion of the sensor and bound to an insulating moiety.
  • SEQ ID NO:78 nucleic acid sensor molecule sensor
  • SEQ ID NO:79 hybridizing to a portion of the sensor and bound to an insulating moiety.
  • Figure 17 is a schematic of the net electron transfer to or from the electrode.
  • Figure 18 is a schematic of a peak in the faradaic current, centered at the redox potential of the electron donor species (specified for a given reference electrode) and superposed on top of the capacitive current baseline which is observed in the absence of surface-immobilized signaling probes.
  • Figure 19 is the sequence of the entire ERK2 activated allosteric ribozyme (SEQ ID NO: 80). Also shown are the sequences of the Stem II connector domain for selective clones.
  • Figure 20A is a chart showing measurement of cis-hammerhead cleavage.
  • 20B shows a chart showing measurement of cis-hammerhead cleavage.
  • Figure 20C is a chart showing measurement of cis-hammerhead cleavage.
  • Figure 21 is a chart showing ERK2-dependence of cis-hammerhead cleavage.
  • Figure 22A is a chart showing the measurement of ERK2-inhibitor IC50 values by nucleic acid sensor molecule.
  • Figure 22B is a chart showing the fraction of construct 1-14 cleaved in the presence of 100 nM ERK2, ricin, or MEK, or with no protein.
  • Figure 23A shows a ppERK cis-hammerhead nucleic acid sensor molecule construct (SEQ ID NO:353).
  • Figure 23B shows construct 6 (SEQ ID NO:81) 7 (SEQ ID NO:82), 8 (SEQ ID NO:
  • Figure 24 shows the linker region, activity, and stability of constructs 6 through 14.
  • Figures 25A and B show a bar graph and corresponding radiograph demonstrating the relative pp ERK dependence of constructs 6, 10, and 12.
  • Figure 26 shows the sequences for lysozyme modulated ligase nucleic acid sensor molecules C.lys.LLA (SEQ ID NO: 105), C.lys.Ll .B (SEQ ID NO: 106), and C.lys.Ll.C (SEQ ID NO: 107).
  • Figure 27 is a schematic of how an LI ligase is configured for self-circularization, and how its self-circularization can be detected using RT-PCR.
  • Figure 28 shows increases in amplification of circularized C.lys.Ll.B (SEQ ID NO: 106) in response to the addition of lysozyme.
  • the signal is strengthened as additional cycles of PCR are performed.
  • Figure 29 shows that the ligase nucleic acid sensor molecule C.lys.Ll .B (SEQ ID NO: 106) still self ligates in response to the presence of lysozyme, even in the presence of HeLa cell extract, demonstrating the stability of this nucleic acid sensor molecule.
  • Figure 30 shows modulation of a 1-piece ligase nucleic acid sensor molecule in vivo.
  • Figure 31 is a schematic showing the construct design for an ERK dependent 3-piece ligase nucleic acid sensor molecule (SEQ ID NO: 1 18).
  • Figure 32 shows the sequences for constructs 17 (SEQ ID NO: 109), 18 (SEQ ID NO:l 10), 19 (SEQ ID NO: 111), 20 (SEQ ID NO:l 12), and 21 (SEQ ID NO:l 13), 22 (SEQ ID NO:114), 23 (SEQ ID NOT 15), 24 (SEQ ID NOT 16), 25 (SEQ ID NOT 17), and 26 (SEQ ID NO: 118).
  • Figure 33 shows the ERK dependent activity of constructs 17 (A), 18 (B), 20 (C), 21 (D), 19 (E), 22 (F), 23 (G), 25 (H), and 26 (I).
  • Figure 34 is a graph that shows ligase time-dependent activity assays for construct 17 (clone A) (SEQ ID NO: 109) and construct 19 (clone E) (SEQ ID NOT 11).
  • Figure 35 shows a graph showing the time-dependent activity of construct 19 (clone
  • Figure 36 shows secondary structure representations of 3-piece construct 27 (SEQ ID NO: 1 18) and 1-piece construct 28 (SEQ ID NOT 19) ERK dependent ligases.
  • 1-piece ERK dependent ligase is a slightly modified version of 3-piece system where the effector and substrate regions are replaced by a stable GNRA tetraloop.
  • Figure 37 shows a graph demonstrating continued ERK2 dependence of a nucleic acid sensor molecule in the 3-piece and 1-piece formats (constructs 19 and 28, respectively).
  • Figure 38 shows a secondary structure representation of the 2-piece ERK dependent ligase platform (SEQ ID NO:347), and its oligonucleotide substrate (SEQ ID NO:350).
  • Figure 39A shows a secondary structure representation of two 2-piece ERK- modulated nucleic acid sensor molecules: construct 29 (SEQ ID NO:349), and construct 30 (SEQ ID NO:351), with their oligonucleotide substrate (SEQ ID NO:348).
  • Figure 39B shows ligation assays run on constructs 29 (SEQ ID NO:349) and 30 (SEQ ID NO:351), in the absence or in the presence of 1 uM ERK.
  • Figure 40 shows the ligation efficiency of ERK nucleic acid sensor molecule construct 19 (SEQ ID NOT 11) detected with quantitiative-PCR (Taqman ® ). All incubation with various concentration of ERK were performed in the presence of 10% 293 extracts with exogenously added 10 mM MgCl 2 .
  • Figure 41 shows a schematic of the template for ppERK dependent ligase nucleic acid sensor molecules (SEQ ID NO:352) and its oligonucleotide substrate (SEQ ID NO:350).
  • Figure 42 shows the nucleotide sequences for construct 31 (TK.16.118.K) (SEQ ID NO:121), construct 32 (TK.16.118.L) (SEQ ID NO:122), construct 33 (TK.16.118.M) (SEQ ID NO: 123), construct 34 (TK.16.1 18.N) (SEQ ID NO: 124), construct 35 (TK.16.118.0) (SEQ ID NO:125), construct 36 (TK.16.118.P) (SEQ ID NOT26), construct 37 (TK.16.118.Q) (SEQ ID NO: 127), construct 38 (TK.16.118.R) (SEQ ID NOT28), construct 39 (TK.16.118.S) (SEQ ID NO: 129), and construct 40 (TK.16.118.T) (SEQ ID NO: 130).
  • Figure 43 shows template sequences for the creation of a ppERK (SEQ ID NO:354) or ERK (SEQ ID NO:355) library of nucleic acid sensor molecules.
  • Figure 44A shows the stem sequences of ERK dependent nucleic acid clones CW45- 33-A08 (SEQ ID NO:356), CW45-33-C08 (SEQ ID NO: 131), CW45-33-C09 (SEQ ID NO: 132), CW45-33-D09 (SEQ ID NO: 133), CW45-33-F08 (SEQ ID NO:90), CW45-33- H08 (SEQ ID NO:91), CW45-33-H09 (SEQ ID NO:92), CW45-33-A10 (SEQ ID NO:93), CW45-33-F09 (SEQ ID NO:94), and CW45-33-G08 (SEQ ID NO:95).
  • Figure 44B shows the stem sequences of pp ERK dependent nucleic acid clones CW45-33-A02 (SEQ ID NO:44), CW45-33-B04 (SEQ ID NO:45), CW45-33-C04 (SEQ ID NO:5), CW45-33-D04 (SEQ ID NO:6), CW45-33-F03 (SEQ ID NO:7), CW45-33-D01 (SEQ ID NO:8), CW45-33-D02 (SEQ ID NO:37), CW45-33-D05 (SEQ ID NO:38), CW45- 33-E01 (SEQ ID NO:39), CW45-33-G02 (SEQ ID NO:96), CW45-33-G03 (SEQ ID NO:44), CW45-33-B04 (SEQ ID NO:45), CW45-33-C04 (SEQ ID NO:5), CW45-33-D04 (SEQ ID NO:6), CW45-33-F03
  • Figure 45 nucleotide sequences of CW45-33-A02 (SEQ ID NO:44), and CW45-33- D04 (SEQ ID NO:6).
  • Figure 46 shows a schematic demonstrating amplicon-dependent nucleic acid amplification (ADNA).
  • Figure 47 shows a schematic describing a mechanism used by nucleic acid sensor molecules to transduce signal and the kinetic constants used to characterize NASMs.
  • Figure 48 shows a graph demonstrating the determination of threshold cycle versus log of target molecule concentration using amplicon-dependent nucleic acid amplification via quantitative PCR analysis.
  • Figure 49 shows a graph demonstrating the determination of threshold cycle versus log of target molecule concentration using amplicon-dependent nucleic acid amplification yia SYBR-green analysis.
  • Figure 50 shows a radiograph demonstrating lysozyme sensitive ligase nucleic acid sensor molecule activity in reticulocyte and HeLa cell extract.
  • Figure 51 shows a radiograph showing that ligase activity is relatively unchanged in the presence of cell lysate and various RNase inhibitors.
  • Figure 52 shows a radiograph of a lysozyme modulated nucleic acid sensor molecule in the presence of human serum.
  • Figure 53 shows a schematic describing rolling circle amplification of an amplicon derived from immobilized trans-acting ligase nucleic acid sensor molecules.
  • Figure 54 shows a schematic describing exponential amplification of an amplicon.
  • Figure 55 shows a schematic describing FRET-based signal generation coupled to nucleic acid synthesis of nucleic acid sensor molecules.
  • Figure 56 shows a schematic describing cellular assays using 1-piece ligase nucleic acid sensor molecules.
  • Figure 57 shows bar graphs plotting the rate of activity of nucleic acid sensors when in the presence of different target molecules in vitro.
  • Panel A shows the rate of activity in the presence of ERK and phosphorylated ERK for construct 19 (ligase E) on the left bar and construct 33 (ligase M) on the right bar.
  • Panel B shows construct 19 and 33 rates of activity in the presence of Ras, MEK, ERK, p38, and ricin.
  • Figure 58 shows a graph describing the activity of an ERK modulated nucleic acid sensor molecule in the presence of 10% 293 cell extract in the left panel.
  • the right panel shows the activity of an ERK modulated nucleic acid sensor molecule in the presence of increasing concentrations of staurosporine. Both panels show data determined using quantitive PCR methods (ADNA).
  • Figure 59 lists the switch factor, dissociation constants, catalytic constant and detection limit for an ERK aptamer in comparison to four ERK dependent ligase nucleic acid sensor molecules.
  • Figure 60A and B show RT-PCR gels, and C and D corresponding bar graphs showing ERK modulation of a nucleic acid sensor molecule in vitro (panels A and C) and in biological extracts (panels B and D).
  • Figure 61 shows a schematic describing optical detection based on the modulation of an intron-derived nucleic acid sensor molecule.
  • Figure 62 shows the original solution-phase cGMP-dependent hammerhead nucleic acid sensor molecule FRET construct (SEQ ID NO: 101) and its effector/capture oligo (SEQ ID NO: 102) from which the solid-phase FRET sensor was derived.
  • the fluorophore (F) and quencher (Q) are FAM and DABCYL, respectively.
  • the donor fluorophore (D) and acceptor fluorophore (A) are FAM and AlexaFluor 568, respectively.
  • Figure 63A shows the surface-immobilized FRET sensor before, and Figure 63B shows after, exposure to the activating target molecule (cGMP), followed by subsequent cleavage and dissociation of the sequence fragment containing the acceptor fluorophore (A).
  • Figure 63 C shows the expected kinetic time course signals and Figure 63 D shows the actual kinetic time course signals observed from these sensors in the presence of various concentrations of target.
  • Figure 64 shows fitted kinetic time course signals observed from the solid-phase FRET sensor constructs in a solution-phase assay.
  • Figure 64A shows a graph that plots the signal observed from the donor fluorophore only in the presence of 200 uM cGMP.
  • Figure 64B shows a graph of the parametric fit to the experimental data shown in Figure 64A, verifying that the rate constant for the solid-phase construct is in fact similar to that for the solution-phase construct under similar conditions.
  • Figures 65A and 65B compare the observed pseudo-first order rate constants from solution- and solid-phase FRET sensor constructs.
  • Figure 65 C, D, and E shows experimental data and constructs for multiplexed detection using solution-phase cGMP and cAMP FRET.
  • Figure 66A shows an endonuclease (hammerhead ribozyme)-based nucleic acid sensor immobilized linked to a gold surface via a thiol linker.
  • Figure 66B shows the fraction of this type of sensor cleaved and dissociated as a function of time in the presence of a fixed concentration of target.
  • Figure 66C shows the signal (image density) from a panel of immobilized sensors prior to their exposure to a target-mixture.
  • Figure 66D shows the signal from the uncleaved sensors after exposure to the mixture of all listed targets, while Figure 66E represents the target-dependent cleavage signal. Specific target-dependent activity of each sensor is seen in each case for this multiplexed assay.
  • Figure 67 shows a schematic diagram of the integrated SPReeta SPR sensor module (Figure 67A), as well as the nucleic acid (hammerhead ribozyme) sensor molecule that is immobilized on the gold SPR layer ( Figure 67B).
  • Figures 67C and 67D show typical realtime data generated by the SPR sensor system during sensor loading and target analyte- induced cleavage, respectively.
  • Figure 68 gives the sequences for three cyclic nucleotide-dependent nucleic acid (hammerhead ribozyme) sensors dependent upon cGMP (SEQ ID NO: 135), cCMP (SEQ ID NO: 136), and cAMP (SEQ ID NO: 137) in a conformation suitable for direct 5 ' surface attachment.
  • the schematic shows the SPR sensor construct intended for direct 5' attachment to a native gold surface via a terminal thiol linker.
  • Figure 69 gives the sequences for three cyclic nucleotide-dependent nucleic acid (hammerhead ribozyme) sensors dependent upon cCMP (SEQ ID NO: 138), cAMP (SEQ ID NO: 103), and cGMP (SEQ ID NO: 139) in a conformation suitable for direct 3' surface attachment.
  • the figure schematic shows the SPR sensor construct intended for direct 3' attachment to a neutravidin surface which has been passively adsorbed onto the gold SPR surface via cysteine residues.
  • Figure 70 gives the sequences for three cyclic nucleotide-dependent nucleic acid (hammerhead ribozyme) sensors dependent upon cCMP (SEQ ID NO:40), cAMP (SEQ ID NO:41), and cGMP (SEQ ID NO:42)in a conformation suitable for indirect surface attachment via a capture oligo.
  • the schematic shows the SPR sensor construct intended for indirect surface attachment via a capture oligo to a neutravidin surface which has been passively adsorbed onto the gold SPR surface via cysteine residues.
  • Figure 71 shows the surface loading ( Figure 71A) and target-dependent cleavage
  • Figure 72 shows a plot of SPR signal (in refractive index units, RIU) vs. time for a typical SPR sensor array assay: surface cleaning (dH 2 0, NaOH, PBS), surface loading of the gold SPR layer with neutravidin (NA), requilibration with PBS (PBS), loading of the surface with biotinylated sensor molecules in PBS(HH+B/PBS), requilibration of sensor surface in assay buffer (HH buff), and addition of target in assay buffer (target HH buff).
  • Figure 73 shows a schematic representation of the secondary structure and components of a ligase-based nucleic acid array sensor.
  • the sensor (SEQ ID NO:75) is shown attached to the chip surface via hybridization to a capture oligo (SEQ ID NO 04), and with an external substrate oligo bearing a fluorescent label already ligated into place.
  • the substrate oligo can be either directly labeled (as shown), or labeled with an affinity tag (e.g., biotin) for subsequent indirect labeling or signal amplification (e.g., via tyramide signal amplification).
  • Figure 74 contrasts the two principal solid-phase array (chip) formats used for ligase-based nucleic acid sensors.
  • Figure 75 shows a multiplex in situ ligase sensor chip, with pre-immobilized radiolabeled sensors activatable by lysozyme (LYS) and FMN.
  • Figure 76 shows dose-response data for ERK-dependent ligase-based nucleic acid sensors using a gel-assay (panel A) and a capture chip (pane IB).
  • Figure 77 shows dose-response data for an in situ ligase-based nucleic acid sensor array populated with ERK-dependent unlabeled ligase sensors in Figure 77A and in Figure 77B the retained ligation signal from each spot in the concentration profile was plotted vs. its corresponding target concentration.
  • Figure 78 shows dose-response data for a ERK-dependent ligase-based nucleic acid sensor capture array in Figure 78A and captured and amplified fluorescent ligation signal for each spot is plotted in Figure 78B vs. its corresponding target concentration.
  • Figure 79 shows the components of a generalized construct for an amplifiable ligase-based nucleic acid sensor molecule.
  • Figure 80 shows a generalized strategy for performing a multiplexed capture chip formatted assay with ligase-based nucleic acid sensors.
  • Figure 81 shows a generalized strategy for performing a highly sensitive capture chip formatted assay with ligase-based nucleic acid sensors.
  • Figure 82 shows a schematic describing multiplexed chip assays.
  • the invention is generally drawn to catalytic NASMs (also know as allosteric ribozymes, aptazymes and the like) and optical nucleic acid sensor molecules that may be used to monitor the presence or concentration of various target molecules.
  • Target molecules include a variety of biologically relevant molecules, such as, for example, proteins (including specific post-translationally modified forms of proteins), peptides, nucleic acids, nucleotides, natural products, metabolites, drugs, toxins, biohazards, and ions.
  • the invention also includes methods by which a change in the activity or conformation of a nucleic acid sensor molecule upon recognition of a specific target molecule can be coupled to a quantifiable, measurable signal.
  • the invention also includes methods which allow one to test the inhibitory activity of one or more compounds simultaneously against one or more enzymes or biochemical targets.
  • Assays can be carried out in a variety of formats, including in vitro biochemical assays, in vitro cellular assays, in vivo cellular assays, in solution, on chips or other substrates, or in vivo animal models. These assays have applications in all phases of drug discovery, including target validation and discovery and development, high throughput screening, biochemical assays, in vitro cellular models and in vivo animal models.
  • Nucleic acid sensor molecules are RNAs, DNAs, RNA/DNA hybrids, or derivatives or analogs of nucleic acids that catalyze a chemical reaction and/or undergo a conformational change upon the recognition of a specific target molecule.
  • Nucleic acid sensor molecule - based assays can be carried out using all catalytic platforms, which include endonucleases, such as the hammerhead ribozyme, the hairpin ribozyme, the HDV ribozyme, and the VS ribozyme; ligases, such as the LI ligase, and the class I-III ligases and; group I and group II self-splicing introns.
  • Catalytic NASMs can be generated or selected by a variety of methods both disclosed herein and known in the art. For examples, see WO98/27104, WO01/96559, and WO 00/26226
  • optical nucleic acid sensor molecules Also disclosed herein are optical nucleic acid sensor molecules and methods making them.
  • optical catalytic NASMs generate a detectable optical signal upon recognition of a target molecule.
  • Optical NASMs are generated from catalytic NASMs by addition of an optical signal generating unit.
  • catalytic NASMs can be used, e.g., to detect target molecules either by generation of an optical signal or an amplicon detectable, e.g., by RT-PCR, size gel purification procedures and any other means of seperating variously sized or conformed nucleic acid molecules.
  • Optical NASMs can be used, e.g., to detect target molecules by generation of an optical signal.
  • Optical signals can be generated by optical NASMs in a number of ways.
  • the signal is an optical signal generated, e.g., by the fluorescence of a fluorescent dye.
  • the signal is an optical signal generated by molecules in close proximity to the nucleic acid sensor molecule whose optical or electrochemical properties are affected by the presence of the target molecule bound nucleic acid sensor molecule.
  • the nucleic acid sensor molecules comprise at least one signaling moiety.
  • the nucleic acid sensor molecules comprise first and second signaling moieties whose optical properties change in response to the binding of a target molecule through changes in the proximity of the first and second signaling moieties. Thus, detection can be direct or indirect.
  • a plurality of nucleic acid sensor molecules are provided, either in solution, or immobilized on a substrate, generating a biosensor.
  • a diagnostic system is provided which comprises at least one biosensor in optical communication with a optical signal detector. Methods of using the diagnostic system are also provided, as well as kits for performing the method.
  • the NASMs are used to detect the presence of target molecules in vivo.
  • oligonucleotide is used interchangeably with the term “nucleic acid” and includes RNA or DNA (or RNA/DNA) sequences of more than one nucleotide in either single strand or double-stranded form.
  • a "modified oligonucleotide” includes at least one residue with any of: an altered internucleotide linkage(s), altered sugar(s), altered base(s), or combinations thereof.
  • target molecule is any molecule to be detected.
  • target molecule refers to, any molecule for which nucleic acid sensor molecule exists or can be generated and can be naturally occurring or artificially created.
  • a "signature target molecule” is a target molecule whose expression is correlatable with a trait.
  • a "diagnostic signature target molecule” is a signature target molecule whose expression is, by itself or in combination with other signature target molecules, diagnostic of a trait.
  • a "pathway target molecule” is a target molecule involved in a biological or metabolic pathway and whose accumulation and/or activity is dependent on other target molecules in the same biological or metabolic pathway, or whose accumulation and/or activity affects the accumulation and/or activity of other target molecules in the same biological or metabolic pathway.
  • a "diagnostic pathway target molecule” is a pathway target molecule whose expression/activity and/or structural properties, by itself or in combination with other pathway target molecules, is diagnostic of a particular trait.
  • a "profiling nucleic acid sensor molecule” is a nucleic acid sensor molecule that recognizes a signature target molecule, a diagnostic signature target molecule, a pathway target molecule, and/or a diagnostic pathway target molecule.
  • a “'biosensor” comprises a plurality of nucleic acid sensor molecules.
  • a “profiling biosensor” comprises a plurality of profiling nucleic acid sensor molecules.
  • a molecule which "naturally binds to DNA or RNA" is one which is found within a cell in an organism found in nature.
  • a “target modulation domain” is the portion of a nucleic acid sensor molecule which recognizes a target molecule.
  • the target modulation domain is also sometimes referred to herein as the "target activation site” or “effector modulation domain”.
  • catalytic domain is the portion of a nucleic acid sensor molecule possessing catalytic activity which is modulated in response to binding of a target molecule to the target modulation domain.
  • linker region is a portion of a nucleic acid sensor molecule by or at which the "target modulation domain” and “catalytic domain” are joined.
  • Linker regions include, but are not limited to oligonucleotides of varying length, baseparring phosphodiester, phosphothiolate, and other covalent bonds, chemical moieties (e.g., PEG), PNA, formacetal, bismaleimide, disulfide, and other bifunctional linker reagents.
  • the linker domain is also sometimes referred to herein as a "connector" or "stem”.
  • a "random sequence” or a “randomized sequence” is a segment of a nucleic acid having one or more regions of fully or partially random sequences.
  • a fully random sequence is a sequence in which there is an approximately equal probability of each base (A, T, C, and G) being present at each position in the sequence.
  • a partially random sequence instead of a 25% chance that an A, T, C, or G base is present at each position, there are unequal probabilities.
  • a fixed region is a nucleic acid sequence which is known.
  • amplifying means any step or process or any combination of steps or processes that increases the amount or number of copies of a molecule or class of molecules.
  • a “catalytic nucleic acid sensor molecule” is a nucleic acid molecule comprising a target modulation domain, a linker region, and a catalytic domain.
  • an Optical nucleic acid sensor molecule is a catalytic nucleic acid sensor molecule wherein the catalytic domain has been modified to emit an optical signal as a result of and/or in lieu of catalysis by the inclusion of an optical signal generating unit.
  • nucleic acid sensor molecule refers to either or both of a catalytic nucleic acid sensor molecule and an optical nucleic acid sensor molecule.
  • signal is a detectable physical quantity, impulse or object.
  • an "optical signal” is a signal the optical properties of which can be detected.
  • an "optical signal generating unit” is a portion of a nucleic acid sensor molecule comprising one or more nucleic acic sequences and/or non-nucleic acid molecular entities, which change optical or electrochemical properties or which change the optical or electrochemical properties of molecules in close proximity to them in response to a change in the conformation or the activity of the nucleic acid sensor molecule following recognition of a target molecule by the target modulation domain.
  • a nucleic acid sensor molecule which "recognizes a target molecule” is a nucleic acid molecule whose activity is modulated upon binding of a target molecule to the TMD a greater extent than it is by the binding of any non-target molecule or in the absence of the target molecule.
  • the recognition event between the nucleic acid sensor molecule and the target molecule need not be permanent during the time in which the resulting allosteric modulation occurs. Thus, the recognition event can be transient with respect to the ensuing allosteric modulation (e.g., conformational change) of the nucleic acid precursor molecule or nucleic acid sensor molecule.
  • an “array” or “microarray” refers to a biosensor comprising a plurality of nucleic acid sensor molecules immobilized on a substrate.
  • a “substrate” refers to any physical supporting surface, whether rigid, flexible, solid, porous, gel-based, or of any other material or composition.
  • an "amplicon” is the sequence of a nucleic acid sensor molecule with ligase activity covalently ligated to an oligonucleotide substrate.
  • a “cleavage substrate” is an oligonucleotide or portion of an oligonucleotide cleaved upon target molecule recognized by a target modulation domain of an endonucleolytic nucleic acid sensor molecule.
  • an "oligonucleotide substrate” is an oligonucleotide that is acted upon by the catalytic domain of a nucleic acid sensor molecule.
  • an "effector oligonucleotide” is an oligonucleotide that base pairs with the effector oligonucleotide binding domain of a nucleic acid sensor molecule with ligase activity.
  • an "effector oligonucleotide binding domain” is the portion of the nucleic acid sensor molecule with ligase activity which is complementary to the effector oligonucleotide.
  • a "capture oligonucleotide” is an oligonucleotide that is used to attach a nucleic acid sensor molecule to a substrate by complementarity and/or hybridization.
  • an "oligonucleotide substrate binding domain” is the portion on the nucleic acid sensor molecule with ligase activity that is complementary to and can base pair with an oligonucleotide substrate.
  • oligonucleotide supersubstrate is an oligonucleotide substrate that is complementary to and can base pair with the oligonucleotide substrate binding domain and to the effector oligonucleotide binding domain of a nucleic acid sensor molecule with ligase activity.
  • the oligonucleotide supersubstrate may or may not carry an affinity tag.
  • oligonucleotide supersubstrate binding domain is the region of a nucleic acid sensor molecule with ligase activity that is complementary to and can base pair with the oligonucleotide supersubstrate.
  • template selection refers to a process performed on a pool of nucleic molecules comprising a target modulation domain, a catalytic domain and an oligonucleotide linker region wherein the linker region is fully or partially randomized.
  • rational design/engineering refers to a technique used to construct nucleic acid sensor molecules in which a non-conserved region of a ribozyme is replaced with a target modulation domain and joined to the catalytic domain of the ribozyme by an oligonucleotide linker region.
  • amplicon dependent nucleic acid amplification refers to a technique by which one can amplify the signal of a nucleic acid sensor molecule by use of standard RT/PCR or Real-Time RT-PCR methods.”
  • switch factor is the enhancement observed in the catalytic activity and/or catalytic initial rate of a nucleic acid sensor molecule upon recognition of a target molecule by the target modulation domain.
  • a "c/s-ligase ribozyme” is a ligase ribozyme that ligates its 3' end to its 5' end.
  • the cz ' s-ligase ribozyme is also referred herein as "1-piece ligase” and is a 1 -component system where oligonucleotide substrate, oligonucleotide substrate binding domain, catalytic domain, effector oligonucleotide and effector oligonucleotide binding domains are fused in the format shown in Figure 39.
  • a "tr ⁇ /w-ligase ribozyme” is a ligase ribozyme that ligates its 5 ' end to the 3' end of an oligonucleotide substrate.
  • a "2-piece ligase” is a 2-component ribozyme.
  • the first component consists of the catalytic domain, the linker region, the target modulation domain, the substrate binding domain and the effector oligonucleotide binding domain.
  • the second component is the oligonucleotide substrate that is complementary to the substrate binding domain and the effector oligonucleotide binding domain. This system follows the format shown in Figure 41.
  • a "3-piece ligase” is a 3-component tr ⁇ r ⁇ -ligase ribozyme.
  • the first component consists of the catalytic domain, the linker, the target modulation domain, the substrate binding domain and the effector oligonucleotide binding domain.
  • the second component is the effector oligonucleotide that is complementary to the effector oligonucleotide binding domain.
  • the third component is the oligonucleotide substrate that is complementary to the substrate binding domain. This system follows the format of the 3- piece ligase platform shown in Figure 39. 1. Generating a Target Specific Nucleic Acid Sensor Molecule
  • Catalytic nucleic acid sensor molecules are selected which have a target molecule-sensitive catalytic activity (e.g., ligation or self-cleavage) from a pool of randomized oligonucleotides.
  • the catalytic NASMs have a target modilation domain to which the target molecule specifically binds and a catalytic domain for mediating a catalytic reaction. Binding of a target molecule to the target modulation domain triggers a conformation change and/or change in catalytic activity in the nucleic acid sensor molecule.
  • an optical nucleic acid sensor molecule is generated whose optical properties change upon binding of a target molecule to the target modulation domain.
  • the pool of randomized oligonucleotides comprises the catalytic site of a ribozyme.
  • a heterogeneous population of oligonucleotide molecules comprising randomized sequences is screened to identify a nucleic acid sensor molecule having a catalytic activity which is modified (e.g., activated) upon interaction with a target molecule.
  • Each oligonucleotide in the population comprises a random sequence and at least one fixed sequence at its 5' and/or 3' end.
  • the fixed sequence comprises at least a portion of a catalytic site.
  • the random sequence is flanked at both ends with fixed sequences.
  • the random sequence portion of the oligonucleotide is about 15- 70 (e.g., 30-40) nucleotides in length and can comprise ribonucleotides and/or deoxyribonucleotides.
  • Random oligonucleotides can be synthesized from phosphodiester- linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art (see, e.g., Froehler, et al, 1986a; 1986b. Oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods (see, e.g., Sood, et al., 1977, and Hirose, et al., 1978). Typical syntheses carried out on automated DNA synthesis equipment yield 10 15 -10 17 molecules. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.
  • random oligonucleotides comprise entirely random sequences; however, in other embodiments, random oligonucleotide can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
  • modified oligonucleotides can be used and can include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof.
  • oligonucleotides are provided in which the P(0)0 group is replaced by P(0)S ("thioate”), P(S)S ("dithioate”), P(0)NR 2 ("amidate”), P(0)R, P(0)OR', CO or CH 2
  • oligonucleotides (“formacetal") or 3 '-amine (-NH-CH -CH -), wherein each R or R' is independently H or substituted or unsubstituted alkyl.
  • Linkage groups can be attached to adjacent nucleotide through an -O-N-, or -S- linkage. Not all linkages in the oligonucleotide are required to be identical.
  • the oligonucleotides comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines.
  • the 2'-position of the furanose residue is substituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.
  • Methods of synthesis of 2'-modified sugars are described in Sproat, et al., 1991; Cotten, et al., 1991; and Hobbs, et al., 1973.
  • 2-fluoro-ribonucleotide oligomer molecules can increase the sensitivity of a nucleic acid sensor molecule for a target molecule by ten-to one hundred-fold over those generated using unsubstituted ribo- or deoxyribooligonucleotides (Pagratis, et al., 1997), providing additional binding interactions with a target molecule and increasing the stability of the nucleic acid sensor molecule's secondary structure(s) (Kraus, et al., 1998; Pieken, et al., 1991; Lin, et al., 1994; Jellinek, et al. 1995; Pagratis, et al., 1997).
  • the random sequence portion of the oligonucleotide is flanked by at least one fixed sequence which comprises a sequence shared by all the molecules of the oligonucleotide population.
  • Fixed sequences include sequences such as hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, SP6, and the like), restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores (described further below), sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest.
  • the fixed sequence is approximately 50 nucleotides in length.
  • the fixed sequence comprises at least a portion of a catalytic site of an oligonucleotide molecule (e.g., a ribozyme) capable of catalyzing a chemical reaction.
  • Catalytic sites are well known in the art and include, e.g. a ligase site (see Figure 2), the catalytic sites of Group I or Group II introns (see, e.g., U.S. Patent Number 5,780,272), the catalytic core of a hammerhead ribozyme (see, e.g., U.S. Patent Number 5,767,263 and U.S.
  • Patent Number 5,700,923, and Figure 5, herein or a hairpin ribozyme (see, e.g., U.S. Patent Number 5, 631,359.
  • Other catalytic sites are disclosed in U.S. Patent Number 6,063,566, Koizumi et al., FEBS Lett. 239: 285-288 (1988), Haseloff and Gerlach, Nature 334: 585-59 (1988), Hampel and Tritz, Biochemistry 28: 4929-4933 (1989), Uhlenbeck, Nature, 328: 596-600 (1987), and Fedor and Uhlenbeck, Proc. Natl. Acad. Sci. USA 87: 1668-1672 (1990)).
  • Nucleic acid sensor molecules are generally selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process.
  • Figure 1 A shows a schematic diagram in which the oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule activatable ligase activity.
  • Figure IB shows the hammerhead nucleic acid sensor molecule selection methodology. Each of these methods are readily modified for the selection of NASMs with other catalytic activities.
  • the ligation reaction involves covalent attachment of an oligonucleotide substrate to the 5'-end of the NASM through formation of a phosphodiester linkage.
  • ligation chemistries can form the basis for selection of NASMs (e.g., oligonucleotide ligation to the 3 '-end, alkylation's (Wilson & Szostak), peptide bond formation (Zhang & Czech), Diels-Alder reactions to couple alkenes and dienes (Seelig 8c Jaschke), etc.).
  • NASMs oligonucleotide ligation to the 3 '-end, alkylation's (Wilson & Szostak), peptide bond formation (Zhang & Czech), Diels-Alder reactions to couple alkenes and dienes (Seelig 8c Jaschke), etc.
  • the chemical functional groups that constitute the reactants in the ligation reaction may not naturally appear within nucleic acids.
  • RNA pool in which one of the ligation reactants is covalently attached to each member of the pool (e.g., attaching a primary amine to the 5 '-end of an RNA to enable selection for peptide bond formation).
  • the oligonucleotide population from which the NASMs will be selected is initially screened in a negative selection procedure to eliminate any molecules which have ligase activity even in the absence of target molecule binding.
  • a solution of oligonucleotides comprising a 5" and 3' fixed sequence (“5'-fixed: random: 3 '-fixed”) is denatured with a 3' primer sequence ("3' prime") (e.g., 200 pM) which binds to at least a portion of the 3' fixed sequence.
  • 3' prime e.g. 200 pM
  • the 5'-fixed:random:3'- fixed sequence is 5'.
  • Ligation buffer e.g., 30 mM Tris HCI, pH 7.4, 600 mM NaCl, 1 mM EDTA, 1% NP-40, 60 mM MgCl 2
  • tag-substrate e.g., Tag-UGCCACU
  • Tags encompassed within the scope include, e.g., radioactive labels, fluorescent labels, a chemically reactive species such as thiophosphate, the first member of a binding pair comprising a first and second binding member, each member bindable to the other (e.g., biotin, an antigen recognized by an antibody, or a tag nucleic acid sequence).
  • the reaction is stopped by the addition of EDTA.
  • the reaction can be terminated by removal of the substrate or addition of denaturants (e.g. urea, formamide).
  • Ligated molecules are removed from pool of selectable molecules (STEP 2), generating a population of oligonucleotides substantially free of ligated molecules (as measured by absence of the tag sequence in the solution).
  • the tag is the first member of a binding pair (e.g., biotin) and the ligated molecules ("biotin-oligonucleotide substrate:5'-fixed:random:3 '-fixed") are physically removed from the solution by contacting the sample to a solid support to which the second member of the binding pair is bound (“S”) (e.g., streptavidin).
  • S solid support to which the second member of the binding pair is bound
  • the eluant collected comprises a population of oligonucleotides enriched for non-ligated molecules (5'- fixed:random:3 '-fixed). This step can be repeated multiple times until the oligonucleotide population is substantially free of molecules having target-insensitive ligase activity.
  • the negative selection step can be configured such that catalysis converts active molecules to a form that blocks their ability to be either retained during the subsequent positive selection step or to be amplified for the next cycle of selection.
  • the oligonucleotide substrate used for ligation in the negative selection step can be synthesized without a capture tag.
  • Target-independent ligases covalently self-attach the untagged oligonucleotide substrate during the negative selection step and are then unable to accept a tagged form of the oligonucleotide substrate provided during the positive selection step that follows.
  • the oligonucleotide substrate provided during the negative selection step has a different sequence from that provided during the positive selection step.
  • PCR is carried out using a primer complementary to the positive selection oligonucleotide substrate, only target-activated ligases will be capable of amplification. .
  • a positive selection phase follows.
  • more 3' primer and tagged oligonucleotide substrate are added to the pool resulting from the negative selection step.
  • Target molecules are then added to form a reacted solution and the reacted solution is incubated at 25 °C for about 2 hours (STEP 3).
  • Target molecules encompassed within the scope include, e.g., proteins or portions thereof (e.g., receptors, antigen, antibodies, enzymes, growth factors), peptides, enzyme inhibitors, hormones, carbohydrates, polysaccharides, glycoproteins, lipids, phospholipids, metabolites, metal ions, cofactors, inhibitors, drugs, dyes, vitamins, nucleic acids, membrane structures, receptors, organelles, and viruses.
  • Target molecules can be free in solution or can be part of a larger cellular structure (e.g., such as a receptor embedded in a cell membrane).
  • a target molecule is one which does not naturally bind to nucleic acids.
  • nucleic acid sensor molecules are selected which are activated by target molecules comprising molecules having an identified biological activity (e.g., a known enzymatic activity, receptor activity, or a known structural role); however, in another embodiment, the biological activity of at least one of the target molecules is unknown (e.g., the target molecule is a polypeptide expressed from the open reading frame of an EST sequence, or is an uncharacterized polypeptide synthesized based on a predicted open reading frame, or is a purified or semi-purified protein whose function is unknown).
  • the target molecule does not naturally bind to nucleic acids
  • the target molecule does bind in a sequence specific or nonspecific manner to a nucleic acid sensor molecule.
  • a plurality of target molecules binds to the nucleic acid sensor molecule. Selection for NASMs specifically responsive to a plurality of target molecules (i.e. not activated by single targets within the plurality) may be achieved by including at least two negative selection steps in which subsets of the target molecules are provided.
  • nucleic acid sensor molecules are selected which bind specifically to a modified target molecule but which do not bind to non-modified target molecules.
  • Targeted modifications include, e.g., post-translational modifications of a protein, such as phosphorylation, ribosylation , methylation (Arg, Asp, N, S, or O-directed), prenylation (e.g., farnesyl, geranylgeranyl, and the like), acetylation, acylation, allelic variations within a protein (e.g., single amino acid changes in a protein) and cleavage sites in a protein.
  • intermediates in a chemical synthesis pathway can be targeted, as well as starting and final products.
  • stereochemically distinct species of a molecules can be targeted.
  • the reacted solution is enriched for ligated molecules (biotin-oligonucleotide substrate: 5'- fixed :random:3'-fixed) by removing non-tagged molecules (5'- fixed:random:3'-fixed) from the solution.
  • the tagged oligonucleotide substrate comprises a biotin tag and ligated molecules are isolated by passing the reacted solution over a solid support to which streptavidin (S) is bound (STEP 4).
  • ligated molecules are identified as nucleic acid sensor molecules and released from the support by disrupting the binding pair interaction which enabled capture of the catalytically active molecules. For example, heating to 95° C in the presence of 10 mM biotin allows release of biotin-tagged catalysts from an immobilized streptavidin support.
  • the captured catalysts remain attached to a solid support and are directly amplified (described below) while immobilized.
  • Multiple positive selection phases can be performed (STEPS 3 and 4). In one embodiment, the stringency of each positive selection phase is increased by decreasing the incubation time by one half.
  • ligation of an oligonucleotide to the active species provides a primer binding site that enables subsequent PCR amplification using an oligonucleotide substrate complementary to the original oligonucleotide substrate.
  • Unligated species do not necessarily need to be physically separated from other species because they are less likely to amplify in the absence of a covalently tethered primer binding site.
  • Selected nucleic acid sensor molecules are amplified (or in the case of RNA molecules, first reverse transcribed, then amplified) using an oligonucleotide substrate primer ("S primer”) (e.g., 5 '- AAAAAATGCACTGGACT-3' (SEQ ID NO:3)) which specifically binds to the litgated oligonucleotide substrate sequence (STEP 5).
  • S primer oligonucleotide substrate primer
  • amplified molecules are further amplified with a nested PCR primer that regenerates a T7 promoter ("T7 Primer”) from the 5' fixed and the litigated oligonucleotide substrate sequence (STEP 6).
  • the oligonucleotide pool may be further selected and amplified to eliminate any remaining unligated sequences (5'-fixed:random:3'- fixed) by repeating STEPS 3-7.
  • any number of amplification methods can be used (either enzymatic, chemical, or replication-based, e.g., such as by cloning), either singly, or in combination. Exemplary amplification methods are disclosed in Saiki, et al., 1985; Saiki, et al., 1988; Kwoh, et al., 1989; Joyce, 1989; and Guatelli, et al., 1990.
  • the 3' primer (3' prime) (see STEP 3 in Figure IA) is included in the ligation mixture, selected nucleic acid sensor molecules may require this sequence for activation. In cases where this is undesirable, the 3' primer may be omitted from the mix.
  • the final nucleic acid sensor molecule can be modified by attaching the 3' primer via a short sequence loop or a chemical linker to the 3' end of the nucleic acid sensor molecule, thereby eliminating the requirement for added primer, allowing 3' primer sequence to self-prime the molecule.
  • nucleic acid sensor molecule begins with the synthesis of a ribozyme sequence on a DNA synthesizer. Random nucleotides are incorporated generating pools of roughly IO 16 molecules. Most molecules in this pool are non-functional, but a handful will respond to a given target and be useful as nucleic acid sensor molecules. Sorting among the billions of species to find the desired molecules starts from the complex sequence pool. Nucleic acid sensor molecule are isolated by an iterative process: in addition to the target-activated ribozymes that one desires, the starting pool is usually dominated by either constitutively active or completely inactive ribozymes. The selection process removes both types of contaminants.
  • the starting library of DNA sequences is generated by automated chemical synthesis on a DNA synthesizer. This library of sequences is transcribed in vitro into RNA using T7 RNA polymerase and subsequently purified. Negative selection incubation. In the absence of the desired target molecule of interest, the RNA library is incubated together with the binding buffer alone. During this incubation, non-allosteric (or non-target activated) ribozymes are expected to undergo cleavage. Size-based Purification. Undesired members of the hammerhead pool, those that are constitutively active in the absence of the target molecule, are removed from the unreacted members by PAGE-chromatography; 7M Urea, 8% acrylamide, IX TBE. Higher molecular weight species are eluted as a single broad band from the gel matrix into TBE buffer, then purified for subsequent steps in the selection cycle.
  • RNA pool is then incubated under identical conditions but now in the presence of the target molecule of interest in binding buffer.
  • RT-PCR amplified DNA is purified and transcribed to yield an enriched pool for a subsequent round of reselection.
  • an oligonucleotide population is screened for a nucleic acid sensor molecule which comprises a target molecule having activatable self-cleaving activity.
  • the starting population of oligonucleotide molecules comprises 5' and 3' fixed regions ("5'-fixed and 3' fixed A- 3'f ⁇ xed B") and at least one of the fixed regions, in this example, the 3' fixed region, comprises a ribozyme catalytic core including a self cleavage site (the junction between 3' fixed A-3 'fixed B).
  • the 5'-fixed: random:3' fixed A-3'-fixed B molecule is GGGCGACCCUGAUGAGCCUGG-N 20-5 o-
  • the population of oligonucleotide molecules comprising random oligonucleotides flanked by fixed 5' and 3' sequences are negatively selected to remove oligonucleotides which self-cleave (i.e., 5'-fixed:random:3'- f ⁇ xed-A molecules) even in the absence of target molecules.
  • the oligonucleotide pool is incubated in reaction buffer (e.g., 50 mM Tris HC1, pH 7.5, 20 mM MgCl 2 ) for 5 hours at 25 °C, punctuated at one hour intervals by incubation at 60 °C for one minutes (STEP 1).
  • the uncleaved fraction of the oligonucleotide population (containing 5'- fixed and 3' fixed A-3'-flxed B molecules) is purified by denaturing 10% polyacrylamide gel electrophoresis (PAGE) (STEP 2).
  • PAGE polyacrylamide gel electrophoresis
  • Target molecule dependent cleavage activity is then selected in the presence of target molecules in the presence of reaction buffer by incubation at 23 °C for about 30 seconds to about five minutes (STEP 3).
  • Cleaved molecules (5'- fixed:random:3'fixed-A molecules) are identified as nucleic acid sensor molecules and are purified by PAGE (STEP 4).
  • Amplification of the cleaved molecule is performed using primers which specifically bind the 5'-f ⁇ xed and the 3'-fixed A sequences, regenerating the T7 promoter and the 3'- fixed B site (STEP 5), and the molecule is further amplified further by RNA transcription using T7 polymerase (STEP 6).
  • the process (STEPS 1-6) is repeated until the nucleic acid sensor precursor population is reduced to about one to five unique sequences.
  • Tags can be attached to the 3 '-fixed B sequence and separation can be based upon separating tagged sequences from non-tagged sequences at STEP 4. Chromatographic procedures that separate molecules on the basis of size (e.g., gel filtration) can be used in place of electrophoresis.
  • One end of each molecule in the RNA pool can be attached to a solid support and catalytically active molecules isolated upon release from the support as a result of cleavage.
  • Alternate catalytic cores may be used. These alternate catalytic cores and methods using these cores are also are encompassed within the scope of the invention.
  • Nucleic acid sensor molecules which combine both cleavage and ligase activities in a single molecule can be isolated by using one or a combination of both of the selection strategies outlined independently above for ligases and endonucleases.
  • the hairpin ribozyme is known to catalyze cleavage followed by ligation of a second oligonucleotide substrate (Berzal-Herranz et al.).
  • Target activated sensor precursors based on the hairpin activity can be isolated from a pool of randomized sequence RNAs prepared as described previously with a sequence of the form 5'-
  • Hairpin-based NASMs can be isolated on the basis of target molecule dependent release of the fragment 5'- GUCCUGUUUGAUGCAUACCGAGUAAGUG-3' (SEQ ID NO:74) in the same way that hammerhead-based NASMs are isolated (e.g.,. target molecule dependent increase in electrophoretic mobility or target molecule dependent release from a solid support).
  • nucleic acid sensor molecules can be selected on the basis of their ability to substitute the 3 '-sequence released upon cleavage for another sequence as described in an target molecule independent manner by Berzal-Heranz et al.
  • the original 3'- end of the NASM is released in an initial cleavage event and an exogenously provided oligonucleotide substrate with a free 5'-hydroxyl is ligated back on.
  • the newly attached 3'- end provides a primer binding site that can form the basis for preferential amplification of catalytically active molecules.
  • Constitutively active molecules that are not activated by a provided target molecule can be removed from the pool by (1) separating away molecules that exhibit increased electrophoretic mobility in the absence of an exogenous oligonucleotide substrate or in the absence of target molecule, or (2) capturing molecules that acquire an exogenous oligonucleotide substrate (e.g., using a 3 '-biotinylated substrate and captured re-ligated species on an avidin column.
  • the group I intron self-splicing ribozymes combine cleavage and ligation activities to promote ligation of the exons that flank it.
  • Group I intron-derived NASMs can be isolated from degenerate sequence pools by selecting molecules on the basis of either one or both chemical steps, operating in either a forward or reverse direction. NASMs can be isolated by specifically enriching those molecules that fail to promote catalysis in the absence of target molecule but which are catalytically active in its presence. Specific examples of selection schemes follow. In each case, a pool of RNAs related in sequence to a representative group I intron (e.g., the Tetrahymena thermophila pre-rRNA intron or the phage T4 td intron) serves as the starting point for selection.
  • a representative group I intron e.g., the Tetrahymena thermophila pre-rRNA intron or the phage T4 td intron
  • Random sequence regions can be embedded within the intron at sites known to be important for proper folding and activity (e.g., substituting the P5abc domain of the Tetrahymena intron, Williams et al.).
  • Intron nucleic acid sensor molecules, in this case, sensitive to thio-GMP can be generated as follows. Ernst step, forward direction The intron is synthesized with a short 5 '-exon. In the negative selection step, a guanosine cofactor is provided and constitutively active molecules undergo splicing. In the positive selection step, the target molecule is provided together with thio-GMP. Molecules responsive to the target undergo activated splicing and as a result acquire a unique thiophosphate at their 5'-termini. Thio-tagged NASMs can be separated from untagged ribozymes by their specific retention on mercury gels or activated thiol agarose columns.
  • the method is performed as described in Green & Szostak.
  • An intron is synthesized with a 5'-guanosine and no 5'-exon.
  • An oligonucleotide substrate complementary to the 5'- internal guide sequence is provided during the negative selection step and constitutively active molecules ligate the substrate to their 5'-ends, releasing the original terminal guanosine.
  • a second oligonucleotide substrate with a different 5 '-sequence is provided together with target in the positive selection step.
  • NASMs specifically activated by the target molecule ligate the second oligonucleotide substrate to their 5'-ends.
  • PCR amplification using a primer corresponding to the second substrate can be carried out to preferentially amplify target molecule sensitive nucleic acid sensor molecules.
  • the method is performed as described in Robertson & Joyce.
  • the intron is synthesized with no flanking exons.
  • pool RNAs are incubated together with a short oligonucleotide substrate under conditions which allow catalysis to proceed.
  • a second oligonucleotide substrate with a different 3 '-sequence is provided together with the sensor target.
  • NASMs are activated and catalyze ligation of the 3 '-end of the second substrate.
  • Reverse transcription carried out using a primer complementary to the 3 '-end of the second substrate specifically selects NASMs for subsequent amplification.
  • nucleic acid sensor molecules Once nucleic acid sensor molecules are identified, they can be isolated, cloned, sequenced, and/or resynthesized using natural or modified nucleotides. Accordingly, synthesis intermediates of nucleic acid sensor molecules are also encompassed within the scope, as are replicatable sequences (e.g., plasmids) comprising nucleic acid sensor precursor molecules and nucleic acid sensor molecules.
  • replicatable sequences e.g., plasmids
  • the nucleic acid sensor molecules identified above through in vitro selection comprise a catalytic domain (i.e., a signal generating moiety), coupled to a target modulation domain, (i.e., a domain which recognizes a target molecule and which transduces that molecular recognition event into the generation of a detectable signal).
  • a target modulation domain i.e., a domain which recognizes a target molecule and which transduces that molecular recognition event into the generation of a detectable signal.
  • the target modulation domain is defined by the minimum number of nucleotides sufficient to create a three-dimensional structure which recognizes a target molecule.
  • the nucleic acid sensor molecules of the present invention use the energy of molecular recognition to modulate the catalytic or conformational properties of the nucleic acid sensor molecule.
  • the selection process as described in detail in the present invention identifies novel nucleic acid sensor molecules through target modulation of the catalytic core of a ribozyme.
  • the in vitro selection procedures described herein are distinct from those previously described for affinity-based aptamer selections (e.g., SELEX) in that we show that selective pressure on the starting population of NASMs (starting pool size is as high as IO 14 to IO 17 molecules) results in nucleic acid sensor molecules with enhanced catalytic properties, but not in enhanced binding properties, Figure 59.
  • the NASM selection procedures place selective pressure on catalytic effectiveness of potential NASMS by modulating both target concentration and reaction time-dependence.
  • NASMs that have high switch factors
  • Figure 57 NASMs that have high specificity
  • the kinetic properties of the NASMs of the present invention are consistent with that obtained from a nucleoprotein-selection reported previously by Robertson and Ellington (2001) in which the resulting ribozyme (switch factor equal to 1,700 fold) has the same affinity for RNA (1 ⁇ M) as did the starting pool.
  • the catalytic site is a known sequence (a ligase site or a hammerhead catalytic core) and is at least a portion of either the 5' and/or 3' fixed region (the other portion being supplied by the random sequence), or is a complete catalytic site.
  • the catalytic site may be selected along with the target molecule binding activity of oligonucleotides within the oligonucleotide pool.
  • the deletion enhances the conformational stability of the optical nucleic acid sensor molecule in either the bound or unbound forms.
  • deletion of the entire catalytic domain of the catalytic NASM shown in Figure 5 is shown to stabilize the unbound form of the nucleic acid sensor molecule.
  • the deletion may be chosen so as to take advantage of the inherent fluorescence-quenching properties of unpaired guanosine (G) residues (Walter, N.G.
  • the target modulation domain from a previously identified nucleic acid sensor molecule is incorporated into an oligonucleotide sequence that changes conformation (e.g., from a duplexed hairpin to a G-quadruplex) upon target binding.
  • Optical Nucleic acid sensor molecules of this type can be derived from allosteric ribozymes, such as those derived from the hammerhead, hairpin, LI ligase, or group 1 intron ribozymes and the like, all of which transduce molecular recognition into a detectable signal.
  • allosteric ribozymes such as those derived from the hammerhead, hairpin, LI ligase, or group 1 intron ribozymes and the like, all of which transduce molecular recognition into a detectable signal.
  • cNMP 3',5'-cyclic nucleotide monophosphate
  • RNA optical nucleic acid sensor molecules which specifically bound to cNMP (Garretta et al., 2001).
  • the catalytic cores for hammerhead ribozymes were removed and replaced with 5-base duplex forming sequences.
  • RNA optical NASMs to c-NMP was then confirmed experimentally.
  • the conformational changes can be coupled to detection via FRET or simply changes in fluorescence intensity (as in the case of a molecular beacon).
  • FRET fluorescence intensity
  • the stabilization of duplex by target binding can be monitored with the change in fluorescence.
  • the above experimental example is performed in solution and utilizes a cuvette-based fluorescence spectrometer, in alternative embodiments the methods are performed in microwell multiplate readers (e.g., the Packard Fusion, or the Tecan Ultra) for high-throughput solution phase measurements.
  • a nucleic acid sensor molecule is bound to a surface by a linker attached to one end of the molecule.
  • a nucleic acid sensor molecule is modified to include a 12 carbon linker terminated with an amine group. This free amine group allows the NASM to be attached to an aldehyde-derivatized glass surface via standard protocols for Schiff base formation and reduction.
  • the nucleic acid sensor molecules can be bound in discrete regions or spots to form an array or uniformly distributed to cover an extended area. In the absence of target molecule, the optical nucleic acid sensor molecule forms a stem-loop conformation with duplex formation along the stem due to the complementarity of the nucleotides at the 3 'and 5' ends of the molecule.
  • the optical nucleic acid sensor molecule undergoes a conformational rearrangement.
  • this conformational rearrangement results in a change in the distance between the fluorophore attached to the 5' end and the quencher attached to the 3' end.
  • the quencher separated from the fluorophore, the detected fluorescence emission intensity from the fluorophore increases sharply.
  • the detected increase in fluorescence intensity with target molecule concentration can be used to detect and quantify the amount of target present in a sample solution introduced onto the surface.
  • a sample solution can be laterally confined about the sensor surface by a coverslip, microwell, incubation chamber seal, or flowcell.
  • an optical signaling unit is either added to, or inserted within, the nucleic sensor molecule, generating a sensor molecule whose optical properties change in response to binding of the target molecule to the target modulation domain.
  • the optical signaling unit is added by exposing at least a 5' or 3' nucleotide that was not previously exposed.
  • the 5' nucleotide or a 5' subterminal nucleotide (e.g., an internal nucleotide) of the molecule is couplable to a first signaling moiety while the 3' nucleotide or 3' subterminal nucleotide is couplable to a second signaling moiety.
  • Target molecule binding to the optical nucleic acid sensor molecule alters the proximity of the 5' and 3' nucleotide (or subterminal nucleotides) with respect to each other, and when the first and second signaling moieties are coupled to their respective nucleotides, this change in proximity results in a target sensitive change in the optical properties of the nucleic acid sensor molecule. Detection of changes in the optical properties of the nucleic acid sensor molecule can therefore be correlated with the presence and/or quantity of a target molecule in a sample.
  • optical NASMs are generated by adding first and second signaling moieties, that are coupled to the 5' terminal or subterminal sequences, and 3 '- terminal and subterminal sequences respectively, of the catalytic NASM.
  • Signaling molecules can be coupled to nucleotides which are already part of the nucleic acid sensor molecule or may be coupled to nucleotides which are inserted into the nucleic acid sensor molecule, or can be added to a nucleic acid sensor molecule as it is synthesized. Coupling chemistries to attach signaling molecules are well known in the art (see, for example, The Molecular Probes Handbook, R. Haughland).
  • Suitable chemistries include, e.g., derivatization of the 5-position of pyrimidine bases (e.g., using 5 '-amino allyl precursors), derivatization of the 5'-end (e.g., phosphoroamidites that add a primary amine to the 5'-end of chemically-synthesized oligonucleotide) or the 3'-end (e.g., periodate treatment of RNA to convert the 3'-ribose into a dialdehyde which can subsequently react with hydrazide- bearing signaling molecules).
  • derivatization of the 5-position of pyrimidine bases e.g., using 5 '-amino allyl precursors
  • derivatization of the 5'-end e.g., phosphoroamidites that add a primary amine to the 5'-end of chemically-synthesized oligonucleotide
  • the 3'-end e
  • a single signaling moiety is either added to, or inserted within, the catalytic nucleic sensor molecule.
  • binding of the target molecule results in changes in both the conformation and physical aspect (e.g., molecular volume, and thus rotational diffusion rate, etc.) of the optical nucleic acid sensor molecule.
  • Conformational changes in the optical nucleic acid sensor molecule upon target binding will modify the chemical environment of the signaling moiety, while changes in the physical aspect of the nucleic acid sensor molecule will alter the kinetic properties of the signaling moiety. In both cases, the result will be a detectable change in the optical properties of the nucleic acid sensor molecule.
  • the optical nucleic acid sensor molecule is prepared without a quencher group. Instead of a quencher group a moiety with a free amine group can be added. This free amine group allows the sensor molecule to be attached to an aldehyde- derivatized glass surface via standard protocols for Schiff base formation and reduction.
  • the nucleic acid sensor molecules can be bound in discrete regions or spots to form an array, or uniformly distributed to cover an extended area.
  • the optical nucleic acid sensor molecule will diffusionally rotate about its point of attachment to the surface at a rate characteristic of its molecular volume and mass. After target binding, the optical NASM-target complex will have a correspondingly larger volume and mass. This change in molecular volume (mass) will slow the rate of rotational diffusion, and result in a measurable change in the polarization state of the fluorescence emission from the fluorophore.
  • a single signaling moiety is attached to a portion of a catalytic NASM that is released as a result of catalysis (e.g., either end of a self- cleaving ribozyme or the pyrophosphate at the 5 '-end of a ligase).
  • a catalytic NASM that is released as a result of catalysis (e.g., either end of a self- cleaving ribozyme or the pyrophosphate at the 5 '-end of a ligase).
  • Target molecule- activated catalysis leads to release of the signaling moiety from the optical NASM to generate a signal correlated with the presence of the target.
  • Release can be detected by either (1) changes in the intrinsic optical properties of the signaling moiety (e.g., decreased fluorescence polarization as the released moiety is able to tumble more freely in solution), or (2) changes in the partitioning of the signaling moiety (e.g., release of a fluorophore from a chip containing immobilized ribozymes such that the total fluorescence of the chip is reduced following washing).
  • changes in the intrinsic optical properties of the signaling moiety e.g., decreased fluorescence polarization as the released moiety is able to tumble more freely in solution
  • changes in the partitioning of the signaling moiety e.g., release of a fluorophore from a chip containing immobilized ribozymes such that the total fluorescence of the chip is reduced following washing.
  • the catalytic nucleic acid sensor molecule is unmodified and the optical signaling unit is provided as a substrate for the NASM.
  • the optical signaling unit is provided as a substrate for the NASM.
  • One example of this embodiment includes a fluorescently tagged oligonucleotide substrate which can be joined to a NASM with ligase activity.
  • analyte-containing samples are incubated with the fluorescent oligonucleotide substrate and the ligase under conditions that allow the ligase to function.
  • the ligase is separated from free oligonucleotide substrate (e.g., by capturing ligases onto a solid support on the basis of hybridization to ligase- specific sequences or by pre-immobilizing the ligases on a solid support and washing i extensively).
  • Quantitation of the captured fluorescence signal provides a means for inferring the concentration of analyte in the sample.
  • catalytic activity alters the fluorescence properties of a oligonucleotide substrate without leading to its own modification.
  • Fluorophore pairs or fluorophore/quencher pairs can be attached to nucleotides flanking either side of the cleavage site of an oligonucleotide substrate for a trans-acting endonuclease ribozyme (Jenne et al.). Target activated cleavage of the substrate leads to separation of the pair and a change in its optical properties.
  • the ligase catalytic NASM and its oligonucleotide substrates are unmodified and detection relies on catalytically-coupled changes in the ability of the NASM to be enzymatically amplified.
  • a target-activated ligase is incubated together with oligonucleotide substrate and an analyte- containing sample under conditions which allow the ligase to function. Following an incubation period, the reaction is quenched and the mixture subjected to RT/PCR amplification using a primer pair that includes the oligo sequence corresponding to the ligation substrate.
  • Amplification products can be detected by a variety of generally practiced methods (e.g. Taqman®).
  • FET fluorescence energy transfer
  • FRET fluorescence resonance energy transfer
  • nonradiative energy transfer long-range energy transfer
  • dipole-coupled energy transfer dipole-coupled energy transfer
  • Forster energy transfer see, e.g., U.S. Patent Number 5,491,063, Wu, and Brand, 1994.
  • proximity-dependent signaling systems that do not rely on direct energy transfer between signaling moieties are also known in the art and can be used in the methods described herein. These include, e.g., systems in which a signaling moiety is stimulated to fluoresce or luminesce upon activation by the target molecule. This activation may be direct (e.g., as in the case of scintillation proximity assays (SPA), via a photon or radionuclide decay product emitted by the bound target), or indirect (e.g., as in the case of AlphaScreenTM assays, via reaction with singlet oxygen released from a photosensitized donor bead upon illumination).
  • SPA scintillation proximity assays
  • AlphaScreenTM assays via reaction with singlet oxygen released from a photosensitized donor bead upon illumination
  • the activation of detected signaling moiety is dependent on close proximity of the signaling moiety and the activating species.
  • the nucleic acid sensor molecule may be utilized in either solution-phase or solid- phase formats. That is, in functional form, the nucleic acid sensor molecule may be tethered (directly, or via a linker) to a solid support or free in solution. In one embodiment, a scintillation proximity assay (SPA) is used.
  • SPA scintillation proximity assay
  • the nucleic acid sensor molecules, ligate on oligonucleotide substrate in the presence of a target molecule are bound to a scintillant-impregnated microwell plate (e.g., FlashPlates, NEN Life Sciences Products , Boston, MA) coated with, for example, streptavidin via a (biotin) linker attached to the 5' end of the effector oligonucleotide sequence (for example, GCGACTGGACATCACGAG (SEQ ID NO:51) in Figure 2A).
  • a scintillant-impregnated microwell plate e.g., FlashPlates, NEN Life Sciences Products , Boston, MA
  • streptavidin e.g., streptavidin
  • a linker attached to the 5' end of the effector oligonucleotide sequence (for example, GCGACTGGACATCACGAG (SEQ ID NO:51) in Figure 2A).
  • the various plate-sensor coupling chemistries are
  • oligonucleotide substrate Upon the addition of a solution containing target molecule and excess radiolabeled (e.g., with 3 p or 35g) oligonucleotide substrate in ligation buffer, the NASMs hybridize and ligate the substrate oligonucleotide. Some fraction of the radiolabeled oligonucleotide substrate will be ligated to surface- immobilized NASMs on the plate, while unligated oligonucleotide substrate will be free in solution.
  • radiolabeled e.g., with 3 p or 35g
  • oligonucleotide substrates ligated to surface-immobilized NASMs on the plate will be in close enough proximity to the scintillant molecules embedded in the plate to excite them, thereby stimulating luminescence which can be easily detected using a luminometer (e.g., the TopCount luminescence plate reader, Packard Biosciences, Meriden, CT).
  • a luminometer e.g., the TopCount luminescence plate reader, Packard Biosciences, Meriden, CT.
  • This type of homogeneous assay format provides straightforward, real-time detection, quantification, and kinetic properties of target molecule binding.
  • a similar SPA assay format is performed using scintillant- impregnated beads (e.g., Amersham Pharmacia Biotech, Inc., Piscataway, NJ).
  • the NASMs which ligate on oligonucleotide substrate in the presence of a target molecule are coupled to scintillant-impregnated beads which are suspended in solution in, for example, a microwell plate.
  • the various bead-sensor coupling chemistries are determined by the type and manufacturer of the beads, and are well-known in the art.
  • the NASMs hybridize and ligate the oligonucleotide substrate.
  • radiolabeled substrate Some fraction of the radiolabeled substrate will be ligated to surface-immobilized NASMs on the beads, while unligated substrate will be free in solution. Only those substrates ligated to surface-immobilized NASMs on the beads will be in close enough proximity to the scintillant molecules embedded in the beads to excite them, thereby stimulating luminescence which can be easily detected using a luminometer (e.g., the TopCount luminescence plate reader, Packard Biosciences, Meriden, CT). In addition to enabling real-time target detection and quantification, this type of homogeneous assay format can be used to investigate cellular processes in situ in real time.
  • a luminometer e.g., the TopCount luminescence plate reader, Packard Biosciences, Meriden, CT.
  • Figures 11 A and B show an exemplary embodiment of a non-isotopic proximity assay based on nucleic acid sensor molecules used in conjunction with AlphaScreenTM beads (Packard Biosciences, Meriden, CT).
  • the nucleic acid sensor molecules which ligate on oligonucleotide substrate in the presence of a target molecule (see Figures 2A and B), are bound to a chemiluminescent compound-impregnated acceptor bead coated with, for example, streptavidin, via a (biotin) linker attached to the 5' end of the effector oligonucleotide sequence (GCGACTGGACATCACGAG (SEQ ID NO:51) in Figure 2A).
  • a chemiluminescent compound-impregnated acceptor bead coated with, for example, streptavidin via a (biotin) linker attached to the 5' end of the effector oligonucleotide sequence (GCGACTGGACATCACGAG (SEQ ID
  • the various bead-sensor coupling chemistries are determined by the type and manufacturer of the beads, and are well-known in the art.
  • the oligonucleotide substrate is coupled to a photosensitizer-impregnated donor bead coated with, for example, streptavidin, via a (biotin) linker attached to the 3' end of the substrate.
  • the donor (substrate) and acceptor (ribozyme) beads and target molecules are then combined in solution in a microwell plate, some of the NASMs hybridize and ligate the oligonucleotide substrate, bringing the donor and acceptor beads into close proximity ( ⁇ 200 nm).
  • the photosensitizer in the donor bead Upon illumination at 680 nm, the photosensitizer in the donor bead converts ambient oxygen into the singlet state at a rate of approximately 60,000/second per bead.
  • the singlet oxygen will diffuse a maximum distance of approximately 200 nm in solution; if an acceptor bead containing a chemiluminescent compound is within this range, i.e., if ligation has occurred in the presence of the target molecule, chemiluminescence at 370 nm is generated.
  • This radiation is immediately converted within the acceptor bead to visible luminescence at 520-620 nm with a decay half-life of 0.3 sec.
  • the visible luminescence at 520-620 nm is detected using a time-resolved fluorescence/ luminescence plate reader (e.g., the Fusion multifunction plate reader, Packard Biosciences, Meriden, CT).
  • a time-resolved fluorescence/ luminescence plate reader e.g., the Fusion multifunction plate reader, Packard Biosciences, Meriden, CT.
  • This type of nonisotopic homogeneous proximity assay format provides highly sensitive detection and quantification of target molecule concentrations in volumes ⁇ 25 microliters for high throughput screening (see Beaudet et al. 2001).
  • Suitable fluorescent labels are known in the art and commercially available from, for example, Molecular Probes (Eugene, Oreg.). These include, e.g., donor/acceptor (i.e., first and second signaling moieties) molecules such as: fluorescein isothiocyanate (FITC) /tetramethylrhodamine isothiocyanate (TRITC), FITC/Texas Red TM Molecular Probes), FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate (PYS) TC, FITC/Rhodamine X (ROX), FITC/tetramethylrhodamine (TAMRA), and others.
  • donor/acceptor i.e., first and second signaling moieties
  • FITC fluorescein iso
  • nonorganic fluorescent labels are known in the art and are commercially available from, for example, Quantum Dot Corporation, Inc. Hayward CA). These include, e.g., donor/ acceptor (i.e., first and second signaling moieties) semiconductor nanocrystals (i.e., 'quantum dots') whose absorption and emission spectra can be precisely controlled through the selection of nanoparticle material, size, and composition (see, for example, Bruchez et al., 1998, Chan and Nie, 1998, Han et al., 2001).
  • donor/ acceptor i.e., first and second signaling moieties
  • semiconductor nanocrystals i.e., 'quantum dots'
  • DABYL dimethyl aminophenylazo benzoic acid
  • EDANS 5-(2'-aminoethyl) aminonaphthalene
  • Figures 3A and B and 6A and B show exemplary optical nucleic acid sensor molecules derived from catalytic nucleic acid molecules ( Figures 2A and B and 5, respectively), according to two embodiments.
  • Figure 3 shows a catalytic nucleic acid sensor molecule obtained from an- oligonucleotide pool in which the catalytic site was a ligase site.
  • Figure 6 shows a catalytic nucleic acid sensor molecule obtained from an oligonucleotide pool in which the catalytic site was a site mediating self-cleavage.
  • a catalytic nucleic acid sensor molecule from which a portion of a ligase site (e.g., the AGUCG sequence at the 3' end of the nucleic acid sensor precursor molecule, as shown in Figure 2) has been removed is coupled to a first signaling moiety (F) at a first nucleotide (1) and to a second signaling moiety (D) at a second nucleotide (2).
  • the first and second signaling moieties molecules are attached to non-terminal sequences.
  • the position of the non-terminal sequences coupled to signaling moieties is limited to a maximal distance from the 5' or 3' nucleotide which still permits proximity dependent changes in the optical properties of the molecule.
  • Coupling chemistries are routinely practiced in the art, and oligonucleotide synthesis services provided commercially (e.g., Integrated DNA Technologies, Coralville, IA) can also be used to generate labeled molecules.
  • the nucleic acid sensor molecule is used, either tethered to a solid support or free in solution, to detect the presence and concentration of target molecules in a complex biological fluid.
  • the first signaling moiety (F) is a fluorescein molecule coupled to the 5' end and the second signaling molecule (D) is a DABCYL molecule (a quenching group) coupled to the 3' end. Because of the nearly complete base pairing of the non-target molecule activated form (see Figure 3B), this is the favored form of the nucleic acid sensor molecule in the absence of the target molecule. When the nucleic acid sensor molecule is not activated by target molecule, the fluorescent group and the quenching group are in close proximity and little fluorescence is detectable from the fluorescent group.
  • target molecule causes a change in the conformation of the optical nucleic acid sensor molecule shown in Figure 3B to that shown in Figure 3A.
  • the first and second signaling moieties (F and D, respectively) are no longer in sufficient proximity for the quenching group to quench the fluorescence of the fluorescent group, resulting in a detectable fluorescent signal being produced upon recognition of the target molecule.
  • the target modulation domain sequence from a previously identified nucleic acid sensor molecule is incorporated into a separate oligonucleotide sequence which changes conformation upon target recognition as shown in Figures 6A and B.
  • nucleic acid sensor molecules of this type can be derived from allosteric ribozymes, such as those derived from the hammerhead, hairpin, LI ligase, or group 1 intron ribozymes and the like (see Soukup et al., 2001, or Hamaguchi et al., 2001), all of which transduce molecular recognition into a detectable optical signal.
  • a self-cleaving ribozyme such as the hammerhead (in this case attached to a solid support via a linker molecule is shown) is labeled with a fluorphore.
  • the labeled NASM in the unactivated state comprises two oligonucleotide including a transacting cleavage substrate which bears a second fluorescent label.
  • the donor fluorophore on one oligonucleotide NASM
  • the acceptor fluorophore on the cleavage substrate
  • the donor fluorophore on one oligonucleotide NASM
  • the acceptor fluorophore on the cleavage substrate
  • minimal fluorescent emission is detected from the donor fluorophore at wavelength 3, ⁇ 3, upon epi- illumination excitation at wavelength 1, ⁇ l.
  • the cleavage fragment of the cleavage substrate bearing the acceptor fluorophore dissociates from the ribozyme-target complex.
  • the donor fluorophore can no longer undergo de-excitation via FRET, resulting in a detectable increase in its fluorescent emission at wavelength 2, ⁇ 2 (see, for example, Singh, et al., 1999; Wu, and Brand, 1994; Walter and Burke, 1997; Walter et al, 1998).
  • the change in the polarization state of the fluorescent emission from the donor fluorophore can be detected/monitored in addition to changes in fluorescent emission intensity (see, for Singh, 2000).
  • the NASMs are free in solution.
  • the acceptor fluorophore attached to the cleavage substrate is replaced by a quencher group.
  • This replacement will also result in minimal fluorescent donor emission at wavelength 2, ⁇ 2, when the NASM is in the unbound state under epi-illumination excitation at wavelength 1, ⁇ l .
  • the cleavage fragments of the cleavage substrate bearing the donor and quencher groups dissociate from the NASM-target molecule complex. Once separated from the quencher, the donor fluorophore will exhibit a detectable increase in its fluorescent emission at wavelength 2, ⁇ 2.
  • the change in the polarization state of the fluorescent emission from the donor fluorophore (due to the increased diffusional rotation rate of the smaller cleavage fragment) can be detected/monitored in addition to changes in fluorescent emission intensity.
  • NASMs are free in solution.
  • the optical configuration is designed to provide excitation via total internal reflection (TIR)-illumination, as shown in Figure 9 C.
  • the donor fluorophore is attached to the NASM body while the quencher is attached to the cleavage substrate.
  • the fluorescent donor emission at wavelength 2, ⁇ 2 will be minimal.
  • the cleavage fragment of the cleavage substrate bearing the quencher group dissociates from the NASM-target module complex.
  • the donor fluorophore Once separated from the quencher, the donor fluorophore will exhibit a detectable increase in its fluorescent emission at wavelength 2, ⁇ 2.
  • the quencher group can be replaced with an acceptor fluorophore.
  • the donor fluorophore is coupled to the cleavage fragment of the cleavage substrate and the acceptor fluorophore or quencher group is deleted.
  • the polarization state of the fluorescent emission from the donor fluorophore will undergo a detectable change due to the difference in the diffusional rotation rates of the surface-bound ribozyme-target complex and the free cleavage fragment.
  • a universal FRET trans-substrate is synthesized for all NASMs derived from self-cleaving allosteric ribozymes.
  • This substrate would have complementary optical signaling units (i.e., donor and acceptor groups) coupled to opposite ends of the synthetic oligonucleotide sequence.
  • Such a universal substrate would obviate the need for coupling optical signaling units to the sensor (i.e., ribozyme) molecule itself.
  • the relative stabilities of the activated and unactivated forms of the nucleic acid sensor molecules is optimized to achieve the highest sensitivity of detection of target molecule.
  • the nucleic acid sensor molecule is further engineered to enhance the stability of one form over another.
  • the boxed UA in Figures 3A and B is changed to a CC, favoring the formation of the target molecule activated form. Because these bases do not form base pairs when the nucleic acid sensor molecule is unactiaved, the unactivated form is not stabilized.
  • any additional proximity dependent signaling system known in the art can be used to practice the method according to the invention, and are encompassed within the scope .
  • the free energy of the structures formed by the nucleic acid sensor molecule is determined using software programs such as mfold®, which can be found on the Rensselaer Polytechnic Institute (RPI) web site (www.rpi.edu/dept.).
  • a gel assay is performed which permits detection of different conformations of the nucleic acid sensor molecule.
  • the nucleic acid sensor molecule is allowed to come to equilibrium at room temperature or the temperature at which the nucleic acid sensor molecule will be used.
  • the molecule is then cooled to 4 °C and electrophoresed on a native (non-denaturing) gel at 4 °C.
  • Each of the conformations formed by the nucleic acid sensor molecule will run at a different position on the gel, allowing visualization of the relative concentration of each conformation.
  • the conformation of nucleic acid sensor molecules which form in the presence of target molecule is then determined by a method such as circular dichroism (CD).
  • CD circular dichroism
  • the conformation which corresponds to the activated conformation can be identified in a sample in which there is no target molecule.
  • the nucleic acid sensor molecule can then be engineered to minimize the formation of the activated conformation in the absence of target molecule.
  • the sensitivity and specificity of nucleic acid sensor molecule can be further tested using target molecule modulation assays with known amounts of target molecules.
  • a catalytical nucleic acid sensor molecule from which a portion of a self-cleaving site has been removed is coupled to a first signaling moiety (F) at a first nucleotide and to a second signaling moiety (D) at a second nucleotide.
  • F first signaling moiety
  • D second signaling moiety
  • the entire catalytic site of the catalytic nucleic acid molecule has been removed.
  • additional bases e.g., UGGUAU
  • bases are selected to be complementary to bases at the opposite end of the nucleic acid sensor molecule (ACCAUA). Additional bases may be added to either the 5' or the 3' end of the nucleic acid sensor molecule.
  • Modifications to stabilize one conformation of the nucleic sensor molecule over another may be identified using the mfold program or native gel assays discussed above.
  • a labeled nucleic acid sensor molecule is generated by coupling a first signaling moiety (F) to a first nucleotide and a second signaling moiety (D) to a second nucleotide as discussed above.
  • F first signaling moiety
  • D second signaling moiety
  • the sensitivity and specificity of the nucleic acid sensor molecule can be further assayed by using target molecule modulation assays with known amounts of target molecules.
  • the optical nucleic acid sensor molecule comprises an optical signaling unit with a single signaling moiety introduced at either an internal or terminal position within the nucleic acid sensor molecule.
  • binding of the target molecule results in changes in both the conformation and physical aspect (e.g., molecular volume or mass, rotational diffusion rate, etc.) of the nucleic acid sensor molecule.
  • Suitable signaling moieties are described in Jhaveri, et al, 2000, and include, e.g., fluorescein, acridine, and other organic and nonorganic fluorophores.
  • a signaling moiety is introduced at a position in the catalytic nucleic acid molecule near the target activation site (identifiable by footprinting studies, for example). Binding of the target molecule will (via a change in conformation of the nucleic acid molecule) alter the chemical environment and thus affect the optical properties of the signaling moiety in a detectable manner.
  • Recognition of the target molecule by the NASM will result in changes in the conformation and physical aspect of the nucleic acid sensor molecule, and will thus alter the kinetic properties of the signaling moiety.
  • the changes in conformation and mass of the sensor-target complex will reduce the rotational diffusion rate for the sensor- target complex, resulting in a detectable change in the observed steady state fluorescence polarization (FP) from the signaling moiety.
  • FP steady state fluorescence polarization
  • concentration can be derived using a modified form of the well-known Michaelis-Menten model for ligand binding kinetics (Lakowicz, 1999).
  • FP is therefore a highly sensitive means of detecting and quantitatively determining the concentration of target molecules in a sample solution
  • FP methods are capable of functioning in both solution- and solid-phase implementations. Numerous additional methods can be used that, e.g., make use of a single fluorescent label and an unpaired guanosine residue (instead of a quencher group), to enable the use of FRET in target detection and quantitation as described in the embodiments above (see Walter and Burke, 1997).
  • an unlabeled ligating ribozyme such as the lysozyme-dependent LI ligase is shown (see, for example, Robertson, M.P. and Ellington, A.D, 2000).
  • TIR total internal reflection
  • oligonucleotide substrates hybridized to NASMs Upon recognition of target molecules in the presence of an oligonucleotide substrate with a tag (where the tag is capable of binding to a subsequently added fluorescent label via interactions including, but not limited to, biotin/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups) those oligonucleotide substrates hybridized to NASMs will undergo ligation and become covalently bonded to the thereto.
  • tag where the tag is capable of binding to a subsequently added fluorescent label via interactions including, but not limited to, biotin/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups
  • oligonucleotide substrate can be added in excess relative to NASM, the temperature of the ambient solution in which the reaction takes place can be kept below room temperature (e.g., 4 °C), and agitation of the reaction vessel can be employed to overcome the kinetic limitation of diffusion-limited transport of species in solution.
  • room temperature e.g. 4 °C
  • agitation of the reaction vessel can be employed to overcome the kinetic limitation of diffusion-limited transport of species in solution.
  • fluorescent label with the appropriate reactive group to bind the substrate tag is added to the reaction mixture. Again, the degree of substrate-label binding can be maximized through control of label concentration, solution temperature, and agitation.
  • the solution temperature can be raised to drive off all of the hybridized but unligated substrate.
  • the spatial extent of the excitation region above the solid substrate surface to which the ribozymes are bound is only on the order of 100 nm. Therefore, the bulk solution above the substrate surface is not illuminated and the detected fluorescent emission will be primarily due to fluorophores which are bound to ligated oligonucleotide substrate-NASM- target molecule complexes tethered to the substrate surface.
  • the fluorescence emission from surface-bound NASM- target molecule complexes in this homogeneous solid phase assay format represents an easily detectable optical signal.
  • the fluorescence polarization (FP) of the labeled substrate can be monitored, as shown in Figure 10 C.
  • the steady state fluorescence polarization signal from the substrate-NASM complex will increase detectably relative to the FP signal from the free labeled oligonucleotide substrate in solution, due to the difference in the diffusional rotation rates between the free and ligated forms.
  • an unlabeled ligating ribozyme such as the lysozyme-dependent LI ligase (see, for example, Robertson, M.P. and Ellington, A.D, 2000) is bound to a solid surface.
  • the oligonucleotide substrate is coupled to an enzyme-linked luminescent moiety, such as horse radish peroxidase (HRP) by a tag (where the tag is capable of binding to a subsequently added label via interactions including, but not limited to, biotin/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups).
  • HRP horse radish peroxidase
  • the activated solution can be precipitated, followed by colorimetric detection.
  • the enzyme linked signal amplification, TSA (sometimes referred to as CARD-catalyzed reporter deposition) is an ultrasensitive detection method.
  • the technology uses turnover of multiple tyramide substrates per horseradish peroxidase (HRP) enzyme to generate high-density labeling of a target protein or nucleic acid probe in situ.
  • Tyramide signal amplification is a combination of three elementary processes: (1) Ligation (or not) of a biotinylated ligase oligonucleotide substrate oligo, followed by binding (or not) of a streptavidin-HRP to the probe; (2) HRP-mediated conversion of multiple copies of a fluorescent tyramide derivative to a highly reactive radical; (3) Covalent binding of the reactive, short lived tyramide radicals to nearby nucleophilic residues, greatly reducing diffusion-related signal loss.
  • Optical nucleic acid sensor molecules for the detection of a target molecule of interest are generated by first selecting catalytic nucleic acid molecules with catalytic activity modifiable (e.g., activatable) by a selected target molecule. In one embodiment, at least a portion of the catalytic site of the catalytic NASM is then removed and an optical signal generating unit is either added or inserted. Recognition of the target molecule by the nucleic acid sensor molecule activates a change in the properties of the optical signaling unit.
  • catalytic activity modifiable e.g., activatable
  • a biosensor which comprises a plurality of optical nucleic acid sensor molecules labeled with first and second signaling moieties specific for a target molecule.
  • the optical NASMs are labeled with a single signaling moiety.
  • the labeled nucleic acid sensor molecules are provided in a solution (e.g., a buffer).
  • the labeled nucleic acid sensor molecules are attached directly or indirectly (e.g., through a linker molecule) to a substrate.
  • nucleic acid sensor molecules can be synthesized directly onto the substrate.
  • Suitable substrates which are encompassed within the scope include, e.g., glass or quartz, silicon, encapsulated or unencapsulated semiconductor nanocrystal materials (e.g., CdSe), nitrocellulose, nylon, plastic, and other polymers.
  • Substrates may assume a variety of configurations (e.g., planar, slide shaped, wafers, chips, tubular, disc- like, beads, containers, or plates, such as microtiter plates, and other shapes).
  • nucleic acid sensor molecules ligate a substrate in the presence of a target molecule (see Figures 2A and B).
  • the ribozymes are bound to a solid substrate via the effector oligonucleotide sequence (for example, GCGACTGGACATCACGAG (SEQ ID NO:51) in Figure 2A).
  • a manual or computer-controlled robotic microarrayer is used to generate arrays of nucleic acid sensor molecules immobilized on a solid substrate.
  • the arrayer utilizes contact-printing technology (i.e., it utilizes printing pins of metal, glass, etc., with or without quill-slots or other modifications).
  • the arrayer utilizes non-contact printing technology (i.e., it utilizes ink jet or capillary-based technologies, or other means of dispensing a solution containing the material to be arrayed).
  • Robotic and manual arrayers are commercially available for example, the SpotArray from Packard Biosciences, Meriden, CT, and the RA-1 from GenomicSolutions, Ann Arbor, MI).
  • larger substrates can be generated by combining a plurality of smaller biosensors forming an array of biosensors.
  • nucleic acid sensor molecules placed on the substrate are addressed (e.g., by specific linker or effector oligonucleotide sequences on the nucleic acid sensor molecule) and information relating to the location of each nucleic acid sensor molecule and its target molecule specificity is stored within a processor.
  • This technique is known as spatial addressing or spatial multiplexing.
  • Techniques for addressing nucleic acids on substrates are known in the art and are described in, for example, U.S. Patent Number 6,060,252, U.S. Patent Number 6,051,380, U.S. Patent Number 5,763,263, U.S. Patent Number 5,763,175, and U.S. Patent Number 5,741,462.
  • nucleic acid sensor molecules are immobilized on a streptavidin-derivatized glass substrate via biotin linkers.
  • the individual sensor spots can be manually arrayed.
  • Solution measurements of target molecule concentration can be made by bathing the surface of the biosensor array in a solution containing the targets (analytes) of interest. In practice this is accomplished either by incorporating the array within a microflowcell (with a flow rate of- 25 microliters/min), or by placing a small volume ( ⁇ 6- 10 microliters) of the target solution on the array surface and covering it with a cover slip.
  • Detection and quantification of target concentration is accomplished by monitoring changes in the fluorescence polarization (FP) signal emitted from the fluorescein label under illumination by 488 nm laser radiation.
  • FP fluorescence polarization
  • the rotational diffusion rate is inversely proportional to the molecular volume; thus the rotational correlation time for the roughly 20-nucleotide unbound sensor (i.e., in the absence of target molecule) will be significantly less than that for the target-NASM complex.
  • the fluorescence emission from the target- NASM complex will therefore experience greater residual polarization due to the smaller angle through which the emission dipole axis of the sensor fluorophore can rotate within its radiative lifetime.
  • different surface attachment chemistries are used to immobilize the NASMs on a solid substrate. As previously noted, these include, e.g., interactions involving biotin/streptavidin, amine/aldehyde, hydrazide, thiol, or other reactive groups
  • the specificity of the biosensors and NASMs according to the invention is determined by the specificity of the target modulation domain of the nucleic acid sensor molecule.
  • a biosensor is provided in which all of the nucleic acid sensor molecules recognize the same molecule.
  • a biosensor is provided which can recognize at least two different target molecules allowing for multi-analyte detection. Multiple analytes can be distinguished by using different combinations of first and second signaling molecules.
  • biosensors may be used to detect multiple analytes using intensity multiplexing. This is accomplished by varying the number of fluorescent label molecules on each biosensor in a controlled fashion.
  • multiple single target biosensors can be combined to form a multianalyte detection system which is either solution-based or substrate-based according to the needs of the user.
  • individual biosensors can be later removed from the system, if the user desires to return to a single analyte detection system (e.g., using target molecules bound to supports, or, for example, manually removing a selected biosensor(s) in the case of substrate-based biosensors).
  • nucleic acid sensor molecules binding to multiple analytes are distinguished from each other by referring to the address of the nucleic acid sensor molecule on a substrate and correlating its location with the appropriate target molecule to which it binds (previously described as spatial addressing or multiplexing).
  • subsections of a biosensor array can be individually subjected to separate analyte solutions by use of substrate partitions or enclosures that prevent fluid flow between subarrays, and microfluidic pathways and injectors to introduce the different analyte solutions to the appropriate sensor subarray.
  • a nucleic acid sensor molecule or biosensor system comprising a nucleic acid sensor molecule in communication with a detector system.
  • a processor is provided to process optical signals detected by the detector system.
  • the processor is connectable to a server which is also connectable to other processors.
  • optical data obtained at a site where the NASM or biosensor system resides can be transmitted through the server and data is obtained, and a report displayed on the display of the off-site processor within seconds of the transmission of the optical data.
  • data from patients is stored in a database which can be accessed by a user of the system.
  • Data obtainable from the biosensors according to the invention include diagnostic data, data relating to lead compound development, and nucleic acid sensor molecule modeling data (e.g., information correlating the sequence of individual sensor molecules with specificity for a particular target molecule).
  • these data are stored in a computer database.
  • the database includes, along with diagnostic data obtained from a sample by the biosensor, information relating to a particular patient, such as medical history and billing information.
  • the database is part of the nucleic acid sensor molecule system, the database can be used separately with other detection assay methods and drug development methods.
  • Detectors used with the nucleic acid sensor molecule systems according to the invention can vary, and include any suitable detectors for detecting optical changes in nucleic acid molecules. These include, e.g., photomultiplier tubes (PMTs), charge coupled devices (CCDs), intensified CCDs, and avalanche photodiodes (APDs).
  • PMTs photomultiplier tubes
  • CCDs charge coupled devices
  • APDs avalanche photodiodes
  • a nucleic acid sensor molecule comprising labeled nucleic acid sensor molecules is excited by a light source in communication with the biosensor.
  • the optical signaling unit comprises first and second signal moieties that are donor/acceptor pairs (i.e., signal generation relies on the fluorescence of a donor molecule when it is removed from the proximity of a quencher acceptor molecule), recognition of a target molecule will cause a large increase in fluorescence emission intensity over a low background signal level.
  • the high signal-to-noise ratio permits small signals to be measured using high-gain detectors, such as PMTs or APDs.
  • Light sources include, e.g., filtered, wide-spectrum light sources, (e.g., tungsten, or xenon arc), laser light sources, such as gas lasers, solid state crystal lasers, semiconductor diode lasers (including multiple quantum well, distributed feedback, and vertical cavity surface emitting lasers (VCSELs)), dye lasers, metallic vapor lasers, free electron lasers, and lasers using any other substance as a gain medium.
  • wide-spectrum light sources e.g., tungsten, or xenon arc
  • laser light sources such as gas lasers, solid state crystal lasers, semiconductor diode lasers (including multiple quantum well, distributed feedback, and vertical cavity surface emitting lasers (VCSELs)), dye lasers, metallic vapor lasers, free electron lasers, and lasers using any other substance as a gain medium.
  • VCSELs vertical cavity surface emitting lasers
  • Common gas lasers include Argon- ion, Krypton-ion, and mixed gas (e.g., Ar-Kr) ion lasers, emitting at 455, 458, 466, 476, 488, 496, 502, 514, and 528 nm (Ar ion); and 406, 413, 415, 468, 476, 482, 520, 531, 568, 647, and 676 nm (Kr ion). Also included in gas lasers are Helium Neon lasers emitting at 543, 594, 612, and 633 nm.
  • Ar-Kr mixed gas
  • Typical output lines from solid state crystal lasers include 532 nm (doubled Nd:YAG) and 408/816 nm (doubled/primary from Ti:Sapphire).
  • Typical output lines from semiconductor diode lasers are 635, 650, 670, and 780 nm.
  • Excitation wavelengths and emission detection wavelengths will vary depending on the signaling moieties used.
  • the excitation wavelength is 488 nm and the emission wavelength is 514 nm.
  • a single excitation wavelength or broadband UV source may be used to excite several probes with widely spectrally separated emission wavelengths (see Bruchez et al., 1998; Chan et al., 1998).
  • detection of changes in the optical properties of the nucleic acid sensor molecules is performed using any of a cooled CCD camera, a cooled intensified
  • the CCD camera a single-photon-counting detector (e.g., PMT or APD), or other light sensitive sensor.
  • the detector is optically coupled to the nucleic acid sensor molecule through a lens system, such as in an optical microscope (e.g., a confocal microscope).
  • a fiber optic coupler is used, where the input to the optical fiber is placed in close proximity to the substrate surface of a biosensor, either above or below the substrate.
  • the optical fiber provides the substrate for the attachment of nucleic acid sensor molecules and the biosensor is an integral part of the optical fiber.
  • the interior surface of a glass or plastic capillary tube provides the substrate for the attachment of nucleic acid sensor molecules.
  • the capillary can be either circular or rectangular in cross-section, and of any dimension.
  • the capillary section containing the biosensors can be integrated into a microfluidic liquid-handling system which can inject different wash, buffer, and analyte-containing solutions through the sensor tube. Spatial encoding of the sensors can be accomplished by patterning them longitudinally along the axis of the tube, as well as radially, around the circumference of the tube interior. Excitation can be accomplished by coupling a laser source (e.g., using a shaped output beam, such as from a VCSEL) into the glass or plastic layer forming the capillary tube.
  • a laser source e.g., using a shaped output beam, such as from a VCSEL
  • the coupled excitation light will undergo TIR at the interior surface/solution interface of the tube, thus selectively exciting fluorescently labeled biosensors attached to the tube walls, but not the bulk solution.
  • detection can be accomplished using a lens- coupled or proximity-coupled large area segmented (pixelated) detector, such as a CCD.
  • a scanning (i.e., longitudinal/axial and azimuthal) microscope objective lens/emission filter combination is used to image the biosensor substrate onto a CCD detector.
  • a high resolution CCD detector with an emission filter in front of it is placed in extremely close proximity to he capillary to allow direct imaging of the biosensors.
  • highly efficient detection is accomplished using a mirrored tubular cavity that is elliptical in cross-section.
  • the sensor tube is placed along one focal axis of the cavity, while a side-window PMT is placed along the other focal axis with an emission filter in front of it. Any light emitted from the biosensor tube in any direction will be collected by the cavity and focused onto the window of the PMT.
  • the optical properties of a nucleic acid sensor molecule are analyzed using a spectrometer (e.g., such as a luminescence spectrometer) which is in communication with the biosensor.
  • a spectrometer e.g., such as a luminescence spectrometer
  • the spectrometer can perform wavelength discrimination for excitation and detection using either monochromators (i.e., diffraction gratings), or wavelength bandpass filters.
  • biosensor molecules are excited at absorption maxima appropriate to the signal labeling moieties being used (e.g., acridine at 450 nm, fluorescein at 495 nm) and fluorescence intensity is measured at emission wavelengths appropriate for the labeling moiety used (e.g., acridine at 495 nm; fluorescein at 515 nm).
  • absorption maxima appropriate to the signal labeling moieties being used
  • fluorescein at 495 nm fluorescein at 495 nm
  • fluorescence intensity is measured at emission wavelengths appropriate for the labeling moiety used (e.g., acridine at 495 nm; fluorescein at 515 nm).
  • the biosensor molecules are in solution and are pipetted (either manually or robotically) into a cuvette or a well in a microtiter plate within the spectrometer.
  • the spectrometer is a multifunction plate reader capable of detecting optical changes in fluorescence or luminescence intensity (at one or more wavelengths), time-resolved fluorescence, fluorescence polarization (FP), absorbance (epi and transmitted), etc., such as the Fusion multifunction plate reader system (Packard Biosciences, Meriden, CT).
  • Fusion multifunction plate reader system Packard Biosciences, Meriden, CT.
  • Such a system can be used to detect optical changes in biosensors either in solution, bound to the surface of microwells in plates, or immobilized on the surface of solid substrate (e.g., a biosensor microarray on a glass substrate).
  • This type of multiplate/multisubstrate detection system coupled with robotic liquid handling and sample manipulation, is particularly amenable to high-throughput, low-volume assay formats.
  • nucleic acid sensor molecules are attached to substrates, such as a glass slide or in microarray format
  • substrates such as a glass slide or in microarray format
  • a small sample volume (-10 nL) is probed to obtain spatial discrimination by using an appropriate optical configuration, such as evanescent excitation or confocal imaging.
  • background light can be minimized by the use of narrow-bandpass wavelength filters between the sample and the detector and by using opaque shielding to remove any ambient light from the measurement system.
  • evanescent wave excitation utilizes electromagnetic energy that propagates into the lower-index of refraction medium when an electromagnetic wave is totally internally reflected at the interface between higher and lower-refractive index materials.
  • a collimated laser beam is incident on the substrate/solution interface (at which the biosensors are immobilized) at an angle greater than the critical angle for total internal reflection (TIR).
  • the substrate is optically coupled (via index-matching fluid) to the upper surface of the prism, such that TIR occurs at the substrate/solution interface on which the biosensors are immobilized.
  • excitation can be localized to within a few hundred nanometers of the substrate/solution interface, thus eliminating autofluorescence background from the bulk analyte solution, optics, or substrate.
  • Target recognition is detected by a change in the fluorescent emission of the nucleic acid sensor, whether a change in intensity or polarization. Spatial discrimination in the plane of the interface (i.e., laterally) is achieved by the optical system.
  • a large area of the biosensor substrate is uniformly illuminated, either via evanescent wave excitation or epi-illumination from above, and the detected signal is spatially encoded through the use of a pixelated detector, such as CCD camera.
  • a pixelated detector such as CCD camera.
  • An example of this type of uniform illumination/CCD detection system (using epi- illumination)) for the case of microarrayed biosensors on solid substrates is the GeneTAC 2000 scanner (GenomicSolutions, Ann Arbor, MI).
  • a small area e.g., 10 x 10 microns to 100 x 100 microns
  • a small area of the biosensor substrate is illuminated by a micro-collimated beam or focused spot.
  • the excitation spot is rastered in a 2-dimensional scan across the static biosensor substrate surface and the signal detected (with an integrating detector, such as a PMT) at each point correlated with the spatial location of that point on the biosensor substrate (e.g., by the mechanical positioning system responsible for scanning the excitation spot).
  • an integrating detector such as a PMT
  • Two examples of this type of moving spot detection system for the case of microarrayed biosensors on solid substrates are: the
  • a small area (e.g., 10 x 10 microns to 100 x 100 microns) of the biosensor substrate is illuminated by a stationary micro-collimated beam or focused spot, and the biosensor substrate is rastered in a 2-dimensional scan beneath the static excitation spot, with the signal detected (with an integrating detector, such as a PMT) at each point correlated with the spatial location of that point on the biosensor substrate (e.g., by the mechanical positioning system responsible for scanning the substrate).
  • an integrating detector such as a PMT
  • An example of this type of moving substrate detection (using confocal epi-illumination) system for the case of microarrayed biosensors on solid substrates is the ScanArray 5000 scanner (Packard Biochip, Billerica, MA).
  • a TIR evanescent wave excitation optical configuration is implemented, with a static substrate and dual-capability detection system.
  • the detection system is built on the frame of a Zeiss universal fluorescence microscope.
  • the system is equipped with 2 PMTs on one optical port, and an intensified CCD camera (Cooke, St. Louis, MO) mounted on the other optical port.
  • the optical path utilizes a moveable mirror which can direct the collimated, polarized laser beam through focusing optics to form a spot, or a beam expander to form a large (> 1cm) beam whose central portion is roughly uniform over the field of view of the objective lens.
  • Another movable mirror can direct the light either to the intensified CCD camera when using large area uniform illumination, or to the PMTs in the scanned spot mode.
  • spot scanning mode a polarizing beamsplitter separates the parallel and perpendicular components of the emitted fluorescence and directs each to its designated PMT.
  • An emission filter in the optical column rejects scattered excitation light from either type of detector.
  • CCD imaging mode manually adjusted polarizers in the optical column of the microscope must be adjusted to obtain parallel and perpendicular images from which the fluorescence polarization or anisotropy can be calculated.
  • a software program interfaces with data acquisition boards in a computer which acquires the digital output data from both PMTs and CCD. This program also controls the PMT power, electromechanical shutters, and galvanometer mirror scanner, calculates and plots fluorescence polarization in real time, and displays FP and intensity images.
  • the detection system is a single photon counter system (see, e.g., U.S. Patent Number 6,016,195 and U.S. Patent Number 5,866,348) requiring rastering of the sensor substrate to image larger areas and survey the different binding regions on the biosensor.
  • the biosensor is used to detect a target molecule through changes in the electrochemical properties of the nucleic acid sensor moleculeor molecules in close proximity to it which occur upon recognition of the NASM to the target molecule.
  • the biosensor system would consist of three major components: One, optical nucleic acid sensor molecules immobilized on an array of independently addressable gold electrodes.
  • the nucleic acid sensor molecules immobilized on each electrode may be modulated by the same or different target molecules, including proteins, metabolites and other small molecules, etc.; two, an oligonucleotide substrate which acts as a signaling probe, hybridizing to the oligonucleotide substrate binding domain of the ligase sensor and forming a covalent phosphodiester bond with the nucleic acid sensor molecule nucleotide adjacent to its 3' terminus in the presence of the appropriate target.
  • This oligonucleotide substrate is typically a nucleic acid sequence containing one or more modified nucleotides conjugated to redox active metallic complexes, e.g., ferrocene moieties, which can act as electron donors; and three, an immobilized mixed self-assembled surface monolayer (SAM), comprised of conductive species separated by insulating species, covering the surface of the electrodes.
  • SAM mixed self-assembled surface monolayer
  • conductive species include thiol- terminated linear molecules, such as oligophenylethyl molecules, while examples of nonconductive thiol-terminated linear molecules, include alkane-thiol molecules terminated with polyethylene glycol (PEG). All immobilized species can be covalently attached to the electrode surface by terminal thiol groups.
  • Figure 15 schematically shows the structure of the mixed self-assembled surface monolayer (SAM) coating the gold electrode, as well as the immobilized nucleic acid sensor molecule (NASM) with a ligated oligonucleotide substrate conjugated to several redox active moieties.
  • SAM mixed self-assembled surface monolayer
  • NAM immobilized nucleic acid sensor molecule
  • the biosensor system would consist of two major components: (1) Optical nucleic acid sensor molecules immobilized on an array of independent addressable gold electrodes.
  • the nucleic acid sensor molecules immobilized on each electrode may be modulated by the same or different target molecules, including proteins, metabolites and other small molecules, etc.
  • the NASM will contain one or more nucleotides conjugated to redox active metallic complexes, e.g., ferrocene moieties, which can act as electron donors; and (2) an immobilized mixed self-assembled surface monolayer (SAM), comprised of conductive species separated by insulating species, covering the surface of the electrodes.
  • SAM mixed self-assembled surface monolayer
  • conductive species include thiol-terminated linear molecules, such as oligophenylehtynyl molecules, while examples of nonconductive thiol- terminated linear molecules include alkane-thiol molecules terminated with polyethylene glycol (PEG).
  • Figure 16 shows the SAM-coated molecule immobilized via a capture oligonucleotide. In this case, the redox active signaling moieties are coupled to the body of the NASM, as shown in the figure.
  • the bulk of the NASM including the nucleotides coupled to the redox active signaling moieties will dissociate from the surface, resulting in a detectable loss of electronic current signal.
  • the array would be subjected, e.g., by an integrated microfluidic flowcell, to an analyte solution containing the target(s) of interest at some unknown concentration.
  • the range of possible sample analyte solutions may include standard buffers, biological fluids, and cell or tissue extracts.
  • the sample solution will also contain the signaling probe at a saturating concentration relative to the immobilized nucleic acid sensor molecule. This ensures that at any given time during analysis, there is a high probability that each nucleic acid sensor molecule will have a signaling probe hybridized to it.
  • the nucleic acid sensor molecule In the presence of the target molecules in the sample solution, the nucleic acid sensor molecule will form a covalent phosphodiester bond, i.e., ligate, with the signaling probe, thus immobilizing it with its redox active electron donor species in electrical contact with the conductive molecules within the mixed self-assembled surface monolayer. After some integration time, during which signal probe ligation occurs, it may be necessary to denature the hybridized but unligated signaling probes.
  • This denaturation step which effectively removes 'background' signaling probes and their associated redox moieties from the vicinity of the electrode, can be accomplished by a small temperature increase (e.g., from 21 °C to 25 °C), or by a brief negative voltage spike applied to the sensor electrodes followed by the application of a large positive DC voltage to a separate electrode that would collect unligated signaling.
  • a sufficiently short hybridization region e.g., 5 base- pairs
  • a separate denaturation step may not be necessary. In either case, following nucleic acid sensor molecule activation by target molecules, a linear electrical potential ramp is applied to the electrodes.
  • the redox species conjugated to the immobilized signaling probe-nucleic acid sensor molecule will be electrochemically oxidized, liberating one or more electrons per moiety.
  • the conductive molecules within the surface monolayer will provide an electrical path for the liberated electrons to the electrode surface, as shown in Figure 17.
  • the net electron transfer to or from the electrode will be measured as a peak in the faradaic current, centered at the redox potential of the electron donor species (specified for a given reference electrode) and superposed on top of the capacitive current baseline which is observed in the absence of surface-immobilized signaling probes. This is shown schematically in Figure 18.
  • Quantitative analysis of the sensor signal is based on the fact that the measured faradaic peak height is directly proportional to number of redox moieties immobilized at the electrode, that is, the number of nucleic acid sensor molecules ligated to signaling probes times the multiplicity of redox moieties per signaling probe molecule. Signal generation by the nucleic acid sensor molecules is thus amplified by virtue of multiple redox species per signaling probe.
  • an alternating current (AC) bias voltage is applied (superposed) on top of the
  • nucleic acid sensor molecule array Multiplexed detection of multiple target molecules simultaneously in a complex sample solution could be accomplished by immobilizing nucleic acid sensor molecules against the target molecules of interest on separate electrodes within a two-dimensional array of electrodes. A complex sample solution containing multiple target molecules and a common signaling probe could then be introduced to the array. All nucleic acid sensor molecules would be exposed simultaneously to all targets, with the target-activated nucleic acid sensor molecule response(s) being observed and recorded only at the spatial location(s) known to contain a nucleic acid sensor molecule specific for the target molecules present in the (unknown) sample. The utility of such a nucleic acid sensor molecule array would be greatly enhanced by the integration of a microfluidic sample and reagent delivery system. Such an integrated microfluidic system would allow the application of reagents and samples to the sensor array to be automated, and would allow the reduction of sample volume required for analysis to ⁇ 1 uL.
  • the sensor array electrodes may be of any configuration, number, and size.
  • the sensor and reference electrodes would be circular gold pads on the order of 100-500 uM in diameter, separated by a center-to center distance equal to twice their diameter. Each electrode would be addressed by separate electrical interconnects.
  • the application of electrical signals to the sensor electrodes can be accomplished using standard commercially available AC and DC voltage sources. Detection of faradaic electrical signals from the sensor electrodes can be accomplished easily using standard commercially available data acquisition boards mounted within and controlled by a microcomputer. Specifically, the raw sensor current signals would need to be amplified, and then converted to a voltage and analyzed via a high resolution (i.e., 16 bit) analog to digital converter (ADC).
  • ADC analog to digital converter
  • an alternating current (AC) bias voltage (at a frequency between, for example, 100 to 1000 Hz) is superposed on top of the DC linear voltage ramp applied to the sensor electrodes.
  • the frequency of the applied bias voltage is called the fundamental frequency.
  • the sensor response signal contains multiple frequency components, including the fundamental frequency and its harmonics (integral multiples of the fundamental frequency).
  • the nth harmonic signal is proportional to the nth derivative of the signal.
  • Detecting these derivative signals minimizes the effects of constant or sloping backgrounds, and can enhance sensitivity by increasing the signal to noise ratio and allowing the separation of closely spaced signal peaks.
  • digital, computer-controlled AC and DC voltage sources i.e., digital to analog converters, DACs
  • current preamplifiers i.e., analog to digital converters (ADCs)
  • ADCs analog to digital converters
  • lock-in amplifiers are all available as integrated signal generation/acquisition boards that can be mounted within and controlled by a single microcomputer.
  • an integrated nucleic acid sensor molecule system with electrochemical detection would include the following elements one, a independently addressable multielement electrode array with immobilized surface layer composed of conductive species separated by insulating species and sensors; two, optical nucleic acid sensor molecules immobilized on the electrode array; three, an oligonucleotide substrate/signaling probe which ligates with the nucleic acid sensor molecule in the presence of the appropriate target; four, an automated or semi-automated microfluidic reagent and sample delivery system; and five, a reader instrument/data acquisition system consisting of a microcomputer controlling the appropriate voltage sources, current and lock- in amplifiers, data acquisition boards, and software interface for instrument control and data collection.
  • the change in activity of the nucleic acid sensor molecule can be detected by watching the change in fluorescence of a nucleic acid sensor molecule when it is immobilized on a chip.
  • a ligase can be attached to a chip and its ligase activity monitored.
  • Ligase ERK nucleic acid sensor molecules, labeled with one fluorophore, e.g., Cy3 is attached via an amino modification to an aldehyde chip.
  • the initial Cy3 fluorescence indicates the efficiency of immobilization of the nucleic acid sensor molecules.
  • the chip is exposed to a substrate labeled with a second fluorophore, e.g., Cy5, with or without the ERK protein target.
  • a second fluorophore e.g., Cy5
  • the nucleic acid sensor molecule ligates the substrate to itself, and becomes Cy5-labeled. Without ERK, the ligation does not occur.
  • an effector oligonucleotide is used to attach the nucleic acid sensor molecule to the chip. Whether one uses effector oligonucleotide or not, the TaqMan (real-time PCR)traces obtained in the presence or absence of target (ERK) for ERK dependent ligases are identical. The presence or absence of this effector oligonucleotide does not affect the activity of the nucleic acid sensor molecule.
  • a hammerhead nucleic acid sensor molecule could be used to measure the concentration of an analyte through the use of fluorescence.
  • Figure 82 shows how many nucleic acid sensor molecules with different effector molecules and/or analytes could be integrated onto one chip to study the concentration of many molecules at once.
  • any optical method known in the art in addition to those described above can be used in the detection and/or quantification of all targets of interest in all sensor formats, in both biological and nonbiological media. These targets include, e.g., those listed in Table 1, below. Any other detection method can also be used in the detection and/or quantification of targets.
  • radioactive labels could be used, including J "P, P, C, H, or I.
  • enzymatic labels can be used including horse radish peroxidase or alkaline phosphatase.
  • the detection method could also involve the use of a capture tag for the bound nucleic acid sensor molecule.
  • the nucleic acid sensor molecules according to the invention can be used to detect virtually any target molecule.
  • the target molecule is a target molecule associated with a pathological condition and detection of changes in the optical properties of the nucleic acid sensor molecules of the biosensor provides a means of diagnosing the condition.
  • Target molecules which are contemplated within the scope include, e.g. proteins, modified forms of proteins, metabolites, organic molecules, and metal ions, as discussed above. Because signal generation in this system is reversible, washing of the biosensor(s) in a suitable buffer will allow the biosensor(s) to be used multiple times, enhancing the reproducibility of the any diagnostic assay since the same reagents can be used over and over.
  • Suitable wash buffers include, e.g., binding buffer without target or, for faster washing, a high salt buffer or other denaturing conditions, followed by re-equilibration with binding buffer.
  • Re-use of the biosensor is enhanced by selecting optimal fluorophores.
  • Alexa Fluor 488 produced by Molecular Probes, has similar optical characteristics compared to fluorescein, but has a much longer lifetime.
  • a site recognized by a nuclease is engineered proximal to the signal generating site, and sequences comprising signaling moieties are removed from the biosensor and replaced by new sequences, as needed.
  • the expression pattern of a plurality of target molecules is determined to obtain a profile of target molecules associated with a trait in an individual to determine an expression pattern which is diagnostic of that trait.
  • combinations of biosensors targeted to individual target molecules are selected until a signature optical profile is determined which is characteristic of a trait.
  • Traits include, e.g., a disease, a genetic alteration, a combination of genetic alterations (e.g., a polygenic disorder), a physiological reaction to an environmental condition, or a wild type state (e.g., of an organism or of an organ system).
  • the target molecules which generate the signature optical profile are identified (based on the type of biosensors used) as signature target molecules.
  • the expression of the signature target molecules can thereafter be determined to identify the presence of the trait in a patient.
  • the expression of the target molecules can be identified using any molecular detection system known in the art; however, in a preferred embodiment, the detection system comprises optical nucleic acid sensor molecules and the trait is identified by detecting the signature optical profile.
  • data relating to the signature optical profile is stored in the memory of a computer.
  • Signature optical profiles can be generated for individual patients or can be generated for populations of individuals. In the latter embodiment, data relating to a composite signature profile (e.g., comprising normalized data) is stored in the memory of a computer or in a computer program product.
  • biosensors according to the invention can be generated which are diagnostic of diseases/traits whose biological basis is not yet known or are the result of complex polygenic interactions and/or of environmental influences.
  • nucleic acid sensor molecules are identified which are activatable by synthetic polypeptides obtained from putative open reading frames identified in the human genome project and/or in other sequencing efforts. Combinations of these activatable nucleic acid sensor molecules (along with activatable nucleic acid sensor molecules specific for target molecules with known functions) are identified which generate a diagnostic optical signal, and signature target molecules are in turn identified which are linked to a particular trait, allowing a biological activity to be associated with a previously uncharacterized molecule.
  • Data relating to signature target molecules or to the optical signals generated upon activation of nucleic acid sensor molecules upon binding to signature target molecules is stored in a database, which can include further information such as sequence information or chemical structure information relating to the signature target molecule.
  • a signature profile relating to a particular trait is generated based on normalized data from a plurality of tests.
  • a signature profile is obtained by determining any or all of the level, chemical structure, or activity, of signature target molecules associated with a disease in samples from a population of healthy individuals to determine a signature profile corresponding to a healthy state.
  • signature profiles are obtained using data from subsets of populations which are divided into groups based on sex, age, exposure to environmental factors, ethnic background, and family history of a disease.
  • methods of drug discovery comprise steps of 1) identifying target(s) molecules associated with a disease; 2) validating target molecules (e.g., mimicking the disease in an animal or cellular model); 3) developing assays to identify lead compounds which affect that target (e.g., such as using libraries to assay the ability of a compound to bind to the target); 4) prioritizing and modifying lead compounds identified through biochemical and cellular testing; 5) testing in animal models; and 6) testing in humans (clinical trials).
  • target activatable nucleic acid sensor molecules according to the present invention offer a way to solve this problem by providing reagents which can be used at each step of the drug development process. Most importantly, the target activatable nucleic acid sensor molecules according to the present invention offer a way to correlate biochemical data, from in vitro biochemistry and cellular assays, with the effect of a drug on physiological response from a biological assay.
  • a method for identifying a drug compound comprising identifying a profile of target molecules associated with a disease trait in a patient, administering a candidate compound to the patient, and monitoring changes in the profile.
  • the monitored profile is compared with a profile of a healthy patient or population of healthy patients, and a compound which generates a profile which is substantially similar to the profile of target molecules in the healthy patient(s) (based on routine statistical testing) is identified as a drug.
  • both the profiling and the drug identification step is performed using at least one nucleic acid sensor molecule whose properties change upon binding to a target molecule.
  • a method for identifying a drug compound comprises identifying a plurality of pathway target molecules, each belonging to a pathway, monitoring the level, chemical structure, and/or activity of pathway target molecules in a patient having a disease trait, administering a candidate compound to the patient, and monitoring changes in the level, chemical structure, and/or activity of the pathway target molecules.
  • the monitored level, chemical structure, and/or activity of the pathway target molecules is compared to the level, chemical structure, and/or activity of pathway target molecules in a wild type patient or patients.
  • both profiling and the identification of drug compounds is performed using at least one nucleic acid sensor molecule whose properties change upon binding to a pathway target molecule.
  • Sensor molecules are target activated optical nucleic acid sensor molecules.
  • Nucleic acid sensor molecules for Use in Identifying Lead Compounds are provided and are validated by testing against multiple patient samples in vitro to verify that the optical signal generated by these molecules is diagnostic of a particular disease. Validation can also be performed ex vivo, e.g., in cell culture, (using microscope-based detection systems and other optical systems as described in U.S. Patent Number 5,843,658, U.S. Patent Number 5,776,782, U.S.
  • the incorporation of biosensors into fiber optic waveguides is known in the art (see, e.g., U.S. Patent Number 4,577,109, U.S. Patent Number 5,037,615, U.S. Patent Number 4,929,561, U.S. Patent Number 4,822,746, and U.S. Patent Number 4,762,799).
  • the selection of fluorescent energy transfer molecules for in vivo use is described in EP-A 649848, for example.
  • nucleic-acid based biosensors are introduced into the body by any suitable medical access device, such as an endoscope or a catheter.
  • the optical fiber is provided within a working lumen of the access device and is in communication with an optical imaging system.
  • the same methods which are used to validate the diagnostic value of particular sets of target molecule/nucleic acid sensor molecule combinations are used to identify lead compounds which can function as drugs.
  • the effects of a compound on target dependent optical signaling is monitored to identify changes in a signature profile arising as a result of treatment with a candidate compound.
  • samples from a treated patient are tested in vitro; however, samples can also be tested ex vivo or in vivo.
  • the diagnostic profile identified by the biosensor changes from a profile which is a signature of a disease to one which is substantially similar to the signature of a wild type state (e.g., as determined using routine statistical tests)
  • the lead compound is identified as a drug.
  • Target molecules which activate the biosensor can comprise molecules with characterized activity and/or molecules with uncharacterized activity. Because large number of target molecules can be monitored simultaneously, the method provides a way to assess the affects of compounds on multiple drug targets simultaneously, allowing identification of the most sensitive drug targets associated with a particular trait (e.g., a disease or a genetic alteration).
  • suitable target molecules include, e.g., nuclear hormone receptor (NHR) polypeptides; G-coupled protein receptor (GPCR) polypeptides, phosphodiesterase (PDE), and protein kinases.
  • NHR nuclear hormone receptor
  • GPCR G-coupled protein receptor
  • PDE phosphodiesterase
  • NHR polypeptides Included in the invention are methods of identifying nucleic acid sensor molecules for detection of conformational isoforms of nuclear hormone receptors, as well as the nucleic acid sensor molecules identified by the methods described herein.
  • Nuclear hormone receptors act as ligand-inducible transcription factors by directly interacting as monomers, homodimers, or heterodimers in complex with DNA response elements of target genes. The activation of these transcription regulators is induced by the change in conformation of the NHR upon complex formation with ligand.
  • the NASMs described herein can include, e.g., those derived from the hammerhead, hairpin, LI ligase or groupl intron ribozymes and the like, any of which transduce molecular recognition into a detectable signal.
  • the mechanistic assays function in both in vitro biochemical as well as with in vitro cell-based settings.
  • the nucleic acid sensor molecules are designed to recognize one conformational isomer of the NHR.
  • the nucleic acid sensor molecule recognizes the unique conformation that exists for the agonist bound form of a hormone receptor; such as that observed for the estrogen receptor ligand binding domain ER-LBD when bound to estrogen (Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL. Cell. 1998;95(7):927-37) and then produces a detectable signal, such as release of fluorescently labeled oligonucleotide, radiolabeled oligonucleotide, or reveals a change in nucleic acid sensor molecule conformation driven by ligand binding through a change in fluorescence or the like.
  • a detectable signal such as release of fluorescently labeled oligonucleotide, radiolabeled oligonucleotide, or reveals a change in nucleic acid sensor molecule conformation driven by ligand binding through a change in fluorescence or the like.
  • the nucleic acid sensor molecule transduces molecular recognition of the ER-LBD-estrogen agonist complex into a detectable signal. The level of the signal is then used to quantify the amount of ER-LBD-estrogen agonist complex present in solution.
  • the ER-LBD-estrogen specific nucleic acid sensor molecule is used as a screening tool in assays designed to detect inhibitors of ER-LBD-estrogen complex formation. These screening tools can be used to determine the inhibition potency of compounds in in vitro biochemical assays or in in vitro cell-based assays.
  • nucleic acid sensor molecules are introduced into cell lines by known methods of electroporation, transfection or coupling to peptide translocating agents such as tat or antennapedia peptides.
  • the ER-LBD-estrogen complex specific nucleic acid sensor molecule is an allosteric intron imbedded in a reporter gene such as GFP or luciferase or the like. When the intron derived nucleic acid sensor molecule is inserted into the reporter gene it renders reporter gene expression effector dependent.
  • functional GFP protein is expressed only when the ER-LBD-estrogen complex is present in the cell, and inhibitors of ER-LBD-estrogen complex formation thus block functional GFP protein expression in appropriate mammalian, such as MCF7 or T47D, yeast or bacterial cell lines.
  • MCF7 or T47D tumor cell lines transfected with GFP-ER- LBD-estrogen nucleic acid sensor molecule sensitive construct are used to form tumor xenografts in nude mice.
  • the transfected tumor xenograft cell lines can be used to form tumors in mice which are not only estrogen dependent but also regulate reporter gene expression in ER-LBD-estrogen dependent manner.
  • NHR ligand binding domains bind antagonists, forming additional conformational isomers.
  • antagonists When antagonists are bound to the receptor a new conformer results such as that observed upon tamoxifen binding to the estrogen receptor to form a stable ER-LBD- tamoxifen complex (Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL. Cell. 1998 Dec 23;95(7):927-37).
  • the invention includes use of an ER-LBD-tamoxifen specific nucleic acid sensor molecule that is used to detect the levels of antagonist specific complex in both in vitro biochemical, cell-based and, in in vivo assays as described above.
  • Nucleic acid sensor molecules can be developed that are specific for the ligand binding domains of all NHRs.
  • nucleic acid sensor molecules for agonist, antagonist, dimeric or multimeric forms of NHR LBDs can be used to screen for inhibitors of LBD function and therefore for inhibitors of NHR dependent transcriptional activation or repression.
  • nucleic acid sensor molecules specific for individual LBD complexes can be used to screen for agents that modify NHR function in in vitro and in in vivo assays.
  • NHRs are multidomain proteins containing a variable NH 2 -terminal region (A/B), a conserved DNA binding domain (DBD) or region (C), a linker region (D), and a conserved region (E) that contains the ligand binding domain (LBD).
  • A/B variable NH 2 -terminal region
  • DBD conserved DNA binding domain
  • C linker region
  • E conserved region
  • NHRs also contain regions required for transcriptional activation, of particular interest is the region AF-2 which is located in the COOH-terminus and whose function is strictly ligand dependent.
  • Provided herein is a method for generating unique nucleic acid sensor molecules to each of the 63 known human NHR LBDs.
  • nucleic acid sensor molecules capable of recognizing the activated state of the NHR by selection for nucleic acid sensor molecules geometries which signal the presence of either the activated or inactivated conformation (NHR with bound ligand), but whose signaling action is quiescent in the presence of other forms of the NHR.
  • the nucleic acid sensor molecules allow the direct, simultaneous, and rapid detection of the activation states of all NHRs.
  • This tool can be used in in vitro assays for receptor activation with agonists and antagonists, and can be used to generate cell lines and animal models that report on the activation state of such receptors in a biological setting and as a function of drug or drug lead.
  • nucleic acid sensor molecules for detection of conformational isoforms of G-protein coupled receptors.
  • GPCRs G-protein coupled receptors
  • GPCRs include three domains: an extracellular N-terminus, a central domain of seven trans-membrane helices connected by unstructured loops, and a cytoplasmic C- terminus. Activation of GPCRs is induced by ligand binding, which causes a conformational change in the receptor transmitting a signal across the plasma membrane to intracellular members of a signaling pathway. The methods described herein allow for generating unique nucleic acid sensor molecules to any GPCR.
  • nucleic acid sensor molecules capable of recognizing the activated state of the GPCR by selecting for nucleic acid sensor molecule geometries which signal the presence or absense of the activated conformation of the receptor through recognition of one or all of the mobile domains, but whose signaling action is quiescent in the presence of other forms of the GPCR.
  • the biosensors described herein include nucleic acid sensor molecules such as allosetric ribozymes (AR), including those derived from the hammerhead, hairpin, LI ligase, or group 1 intron ribozymes and the like. Nucleic acid sensor molecules may also be derived from aptamer beacons or signaling aptamers which transduce molecular recognition into a detectible signal.
  • AR allosetric ribozymes
  • G-protein coupled receptor(GPCR) polypeptides activate intracellular G ⁇ -protein.
  • GPCR G-protein coupled receptor
  • a single GPCR can activate a number of G ⁇ -proteins.
  • adrenergic receptors activate Gi, which inhibit adenylyl cyclases, Gs, which stimulate adenylyl cyclases, and Gq, which regulate cellular Calcium ion level (Wenzel-Seifert and Seifert 2000).
  • Gi adrenergic receptors
  • Gs which stimulate adenylyl cyclases
  • Gq which regulate cellular Calcium ion level
  • the initial drug screening of the GPCRs is normally performed by competition assay with radiolabeled ligands.
  • incorporation of radiolabeled GTP can be measured to detect the coupling of G ⁇ -protein and GPCR, however this assay does not distinguish the type of G ⁇ -proteins involved.
  • the assays for the effect on individual effectors, such as the Ca flow or cellular cAMP level, are also commonly used, but only one downstream signal can be measured at a time using these assays.
  • G ⁇ -protein Upon activation, a G ⁇ -protein undergoes significant conformational change which results in release of GDP and association with GTP (Coleman and Sprang 1998). The G ⁇ - protein also dissociates from its ⁇ -subunits. This activated form of G ⁇ -protein then becomes capable of interacting with its effector (Li, Stern Stamm et al. 1998). The well- characterized conformation change takes place in three switches; switch I (residues 177-187 in Gi ⁇ l), switch II (residues 199-219 in Gi ⁇ l), and switch III (residues 231-242 in Gi ⁇ l). The sequences and the conformational changes in these switches are well conserved among G ⁇ -proteins.
  • Ras is a member of the small GTPase protein family, which shares significant similarity with other family members.
  • GTP-bound ras and GDP-bound ras can be distinguished by the RBD (ras binding domain) of Raf-1 (Taylor, Resnick et al. 2001).
  • the activated state of Raf-1 can be identified by RalRDS (Franke, Akkerman et al. 1997). This indicates significant change in the surface of the protein, and the effector binding surface are only available for interaction in GTP complex form.
  • the invention provides methods for selecting nucleic acid sensor molecules which recognize the conformational change upon GTP binding and/or specifically interact with newly exposed G-protein effector binding sites upon the activation.
  • Class-specific activated G ⁇ -protein nucleic acid sensor molecules recognize the activated G ⁇ -proteins or its effector binding site, which allow us to interpret the multiple type of downstream signal affect by the GPCR. It can used in both in vitro HTS (high throughput screening) and cell-based HTS.
  • Also described is a method for developing a direct mechanistic assay of the action of small molecule ligand-agonism, -antagonism, and partial antagonism of members of the GPCR family.
  • the mechanistic assays function in both in vitro biochemical and in vitro cell-based settings. In the in vitro assay setting, the nucleic acid sensor molecules are designed to recognize one conformational isomer of the GPCR.
  • the nucleic acid sensor molecule recognizes the unique conformation that exists for the activated state when in complex with ligand, e.g., such as that observed for the beta-2 adrenergic receptor when in complex with the artificial ligand isoproterenol (Ghanouni et al., PNAS USA, 98:5997-6002(2001)) and then produces a detectable signal, e.g., by release of a fluorescently labeled oligonucleotide, release of a radiolabeled oligonucleotide, or a change in conformation of the NASM driven by ligand binding through a change in fluorescence or the like.
  • ligand e.g., such as that observed for the beta-2 adrenergic receptor when in complex with the artificial ligand isoproterenol (Ghanouni et al., PNAS USA, 98:5997-6002(2001)
  • a detectable signal e.g.,
  • the nucleic acid sensor molecule transduces molecular recognition of the beta-2 adrenergic receptor — in complex with epinephrine, norepinephrine or an artificial ligand such as isoproterenol into a detectible signal. The level of the signal is then used to quantify the amount of beta-2 adrenergic receptor-agonist complex present in solution.
  • the beta-2 adrenergic-agonist nucleic acid sensor molecule is used as a screening tool in assays designed to detect agonists of the beta-2 adrenergic receptor. These screening tools can be used to determine the activation potency of compounds in in vitro biochemical assays or in in vitro cell-based assays. Agonists of the beta-2 receptor are useful in the treatment of asthma (Robinson, et al. Lancet 357:2007- 201 1(2001)).
  • nucleic acid sensor molecules are introduced into cell lines by known methods of electroporation, transfection, or coupling to peptide translocating agents such as tat or antennapedia peptides.
  • the beta-2 adrenergic receptor-agonist complex specific nucleic acid sensor molecule is an allosteric intron imbedded in a reporter gene such as GFP or luciferase or the like.
  • a reporter gene such as GFP or luciferase or the like.
  • the intron-derived reporter When the intron-derived reporter is inserted into the reporter gene it renders reporter gene expression effector dependent.
  • functional GFP protein is expressed only when the beta-2 adrenergic receptor-agonist complex is present in the cell, and inhibitors of beta-2 adrenergic receptor-agonist complex formation block functional GFP protein expression in appropriate cells such as mammalian human peripheral blood leukocytes, yeast, insect, or bacterial cell lines.
  • Chinese hamster fibroblasts which do not express beta- adrenergic receptors (Sheppard, et al, PNAS USA 80:233-236(1983)), are transfected with both the nucleic acid sensor molecule and the gene coding for the beta-adrenergic receptor under a constitutive promoter, and are used to create a model cell line suitable for HTS screening of candidate beta-2 agonists.
  • cells can be caused to express known allelic variants, such as gln27-to-glu associated with obesity (Large, et al., J Clin. Invest 100:3005-3013), to create cells lines which model specific disease states.
  • chimeric mice can be created by "knock-in” (Monroe et al., Immunity 11:201-212(1999)) which will express the nucleic acid sensor molecule in every cell as the result of blastocyst fusion (Chen et al., PNAS USA 90:4528-4532(1993)), and used for pharmokinetic or bioavailability studies in which the GPCR activation states of various tissues in the organism are of concern.
  • GPCRs bind antagonists, which cause the GPCRs to become resistant to conformational changes, or result in conformations not susceptible to activation, or blockade the ligand binding domain from interaction with the appropriate ligand and thus prevent activation of the GPCR, such as the beta-2 adrenergic receptor antagonist butoxamine (Horinouchi et al., Pharmacology 62:98-102(2001)).
  • the invention also provides a method for using a nucleic acid sensor molecule to detect conformers which result from binding of GPCRs to antagonists.
  • the nucleic acid sensor molecule can be employed in a screen for compounds which are beta-2 antagonists (Ramsay et al, Br J Pharmacol 133:315- 323(2001)). Antagonists of the beta-2 receptor are useful in the treatment of cardiovascular diseases (Nagatomo, et al., Cardiovasc Drug Rev 19:9-24(2001)).
  • the invention accordingly provides a method for using a Beta-2 adrenergic receptor - butoxamine complex-specific nucleic acid sensor molecule that is used to detect the levels of an antagonist specific complex in both in vitro biochemical, cell-based, and in vivo assays as described above.
  • Nucleic acid sensor molecules can also be developed that are specific for the occupancy state of the ligand-binding domains of all GPCRs.
  • nucleic acid sensor molecules for the agonist, antagonist, dimeric, or multimeric forms of all GPCRs can be used to screen for inhibitors or activators of GPCR function and therefore for inhibitors or activators of GPCR-dependent cell signaling pathways.
  • Nucleic acid sensor molecules specific for individual GPCR complexes can be additionally used to screen for agents that modify GPCRs in in vitro and in vivo assays. Phosphodiesterase-specific nucleic acid sensor molecules
  • PDEs phosphodiesterases
  • the invention provides multiple classes of PDE nucleic acid sensor molecules.
  • the first class of nucleic acid sensor molecules can distinguish cAMP vs. 5'AMP (cGMP vs 5'GMP) (Koizumi, Kerr et al. 1999) (Koizumi, Soukup et al. 1999).
  • the second class of nucleic acid sensor molecule binds to the active site of PDE in a class specific manner and inhibits PDE catalytic activity. This class of nucleic acid sensor molecule can be raised using PDEs in the presence and absence of high affinity known inhibitors (e.g., Ropalim for PDE4).
  • the third class of nucleic acid sensor molecule recognizes PDE in a class-specific (e.g. , PDE 1 - 11) or subclass-specific (PDE4A-D) manner.
  • Protein kinase-specific nucleic acid sensor molecules Protein kinase-specific nucleic acid sensor molecules
  • the invention also provides nucleic acid sensor molecules raised against protein kinases.
  • the invention provides nucleic acid sensor molecules that are modulated by the phosphorylation state in a given peptide sequence.
  • native proteins can be used with different phosphorylation states in order to raise nucleic acid sensor molecules that can distinguish the different phosphorylation states.
  • ERKl/2 and phosphorylated ERKl/2 can be distinguished by specific nucleic acid sensor molecules (Seiwert, Stines Nahreini et al. 2000).
  • the nucleic acid sensor molecule also can be a competitive inhibitor for a kinase by binding at ATP or substrate binding sites.
  • an ADP-dependent nucleic acid sensor molecule can be obtained at lower pH. These nucleic acid sensor molecules can be used to detect the production of ADP. ii. Pathway Profiling Biosensors
  • physiological function is modulated by complex pathways, each of which may have multiple overlapping and intersecting steps. Furthermore, the proteins involved in these pathways are highly homologous and can have overlapping substrates and drug specificities. Using current techniques, it is possible only to monitor the response of single elements of a pathway. These techniques are inadequate to understand the mechanism of drug interactions. For example, a particular drug found to have a particular in vitro activity against a single target in biochemical assays might interact with other proteins in the same pathway, or in other unrelated pathways. Consequently, physiological function is often uncorrelated with the results of biochemical assays of a single target.
  • the nucleic acid sensor molecules according to the invention provide reagents to simultaneously quantify the level and chemical state of all components in a molecular pathway
  • path target molecules are target molecules involved in the same pathway and whose accumulation and/or activity is dependent on other pathway target molecules, or whose accumulation and/or activity affects the accumulation and/or activity of other pathway target molecules.
  • Pathway target molecules include, e.g., proteins, such as enzymes, modified forms of proteins, such as phosphorylated, sulfated, ribosylated proteins, methylated proteins (Arg, Asp; N, S or O directed), prenylated proteins (such as by farnesyl, geranylgeranyl, and other types of prenylation) acetylated or acylated proteins, cleaved or clipped proteins, bound or unbound forms of proteins, allelic variants of a protein (e.g, proteins differing from each other by single amino acid changes in a protein), as well as substrates, intermediates, and products of enzymes (including both protein and non-protein molecules).
  • proteins such as enzymes, modified forms of proteins, such as phosphorylated, sulfated, ribosylated proteins, methylated proteins (Arg, Asp; N, S or O directed), prenylated proteins (such as by farnesyl, geranylgeranyl,
  • diagnostic pathway target molecules are identified by preselecting a plurality of nucleic acid sensor molecules activatable by pathway-specific target molecules.
  • a profiling biosensor is provided comprising at least one nucleic acid sensor molecule specific for every molecular species within a pathway (e.g., a signaling pathway), to generate a biosensor which can monitor the levels, chemical structure, and/or activity of every molecular species in the pathway.
  • the profiling biosensors of the instant invention make it feasible to evaluate the response of all the components of a pathway to a drug compound simultaneously.
  • a profiling biosensor reactive to the components of an entire pathway is contacted with a sample from a patient having a disease, and an optical signal corresponding to a disease state is determined to identify diagnostic pathway target molecules which are diagnostic of that disease.
  • Samples from a plurality of patients are obtained and tested using the profiling biosensor to identify a pathway profile that is diagnostic of the disease, the pathway profile comprising normalized data relating to any or all of the level, structure, and activity, of the signature pathway molecules.
  • a pathway profile corresponding to a wild type state is determined by testing the profiling biosensor molecules against samples from a population of healthy patients, or subsets of populations of healthy patients.
  • data relating to the optical signals generated by nucleic acid sensor molecules activated by the diagnostic pathway target molecules is stored within the memory of a computer or within a computer program product.
  • the pathway profiles can be used in diagnostic testing as discussed above.
  • a profiling biosensor is used in which the pathway is one which is known or suspected of being disrupted in patients having a particular trait (e.g., having a particular disease or genetic alteration(s)).
  • one profiling biosensor used to evaluate samples from a patient with cardiovascular disease is a cholesterol metabolism pathway profiling biosensor.
  • random combinations of profiling biosensors can be used to assess the physiological state of a patient, to identify diagnostic pathway profiles which are diagnostic of diseases whose molecular basis has not yet been identified or characterized.
  • profiling biosensors are used to assess the affect of a candidate drug on any or all of the level, chemical structure, or activity of diagnostic pathway target molecules to generate a drug treatment pathway profile.
  • a profiling biosensor is contacted with a sample from a cell or physiological system (e.g., a group of cells, a tissue system, an organ system, or a patient), and changes in optical signals are obtained which are correlated to any, or all of, the level, chemical structure, or activity of a particular pathway target molecule by relating the optical signal obtained to the address of the nucleic acid sensor molecule, as described above.
  • a cell or physiological system e.g., a group of cells, a tissue system, an organ system, or a patient
  • a drug treatment profile which is substantially similar to a diagnostic pathway profile obtained from a healthy population of patients (as determined by observing no significant differences in the profile by routine statistical testing, to within 95% confidence levels) is used to identify a candidate drug as one which is suitable for further testing.
  • the profile produced by such a drug is used to produce an effective drug treatment profile, against which other candidate drugs can be compared.
  • a candidate drug is tested against a plurality of profiling biosensors including the one which will generate a diagnostic signature profile, to identify drugs which produce an effective drug treatment profile without effecting significant 82
  • the systemic effects of a candidate drug can be predicted.
  • a biosensor representing less than an entire pathway.
  • a biosensor comprising nucleic acid sensor molecules diagnostic pathway target molecules.
  • a biosensor which comprises nucleic acid sensor molecules necessary to evaluate particular components of a pathway suspected of being involved in a disease. For example, compounds being screened to identify candidate drugs that affect diseases relating to defective DNA repair can be tested against a pathway biosensor comprising only S phase cell cycle target molecule reactive nucleic acid sensor molecule.
  • Exemplary pathway target molecules include various proteins and modified form thereof. Modifications include post-translational modifications such as phosphorylation, prenylation, glycosylation, methionine removal, N-acetylation, acylation, acylation of cysteines, myristoylation, alkylation, ubiquitinylation, prolyl-4-hydroxylation, carboxylation of glutaminyl residues, advanced glycoslylation (e.g., of hemoglobin), deamination of glutamine and asparagine, addition of glycophosphatidyl inositol, disulfide bond formation, hydroxylation, and lipidation.
  • post-translational modifications such as phosphorylation, prenylation, glycosylation, methionine removal, N-acetylation, acylation, acylation of cysteines, myristoylation, alkylation, ubiquitinylation, prolyl-4-hydroxylation, carboxylation of glutaminyl residues, advanced glycoslylation (e.
  • proteins include ERK and phosphorylated ERK, CDK and phosphorylated CDK, modified cyclin A, cyclin D, cyclin E, k-Ras, h-Ras, Rho A, MEK-1, MEKK-1, Raf-1, Raf-A, JNK, PKA, ATK, PTEN, p53, PI 6, and INK4.
  • Other proteins include those listed in Table 1 as well as post-translationally modified forms thereof.
  • nucleic acid sensor molecules can be raised against particular amino acid sequences in the polypeptides.
  • Some representative peptide regions are presented in Table
  • a profiling biosensor array is generated comprising target activatable nucleic acid sensor molecules which are activatable by components of a cell cycle pathway.
  • a cell cycle biosensor is generated comprising nucleic acid nucleic acid sensor molecules activatable by at least two members selected from the group consisting of: MPS, Cytostatic factor (CSF) (including Mos), cdk4, cyclins Dl-3, cdk6, cdk2, cyclin E, p53, p21, pl6, Rb, p27, E2F, cyclin A, cyclin B, cdkl, cyclin Bl-3, Cdc2, SPA-1, and other biomolecules involved in cell cycle regulation.
  • CSF Cytostatic factor
  • the cell cycle biosensor array generated is used to evaluate samples from patients suspected of having a disorder affecting cell proliferation (e.g., cancer) and a signature target molecule profile is determined which is diagnostic of this disorder. Changes in the signature target molecule profile upon treatment with a candidate compound are subsequently monitored by any or all of in vitro, ex vivo, and in vivo methods, as described above, to identify and/or validate lead compounds for use in cancer therapies.
  • a disorder affecting cell proliferation e.g., cancer
  • a cell cycle biosensor comprising a plurality of locations, each location comprising a set of nucleic acid sensor molecules activatable by target molecules which identify a different portion of the cell cycle.
  • a cell cycle biosensor comprises at a first location, nucleic acid sensor molecules activatable by GO specific target molecules (e.g., MPS, Cytostatic factor (CSF) (including Mos)), at a second location, nucleic acid sensor molecules activatable by G 1 specific target molecules (cdk4, cyclin Dl-3, cdk6, cdk2, cyclin E, p53, p21, pl6, Rb, p27, E2F), at a third location, nucleic acid sensor molecules which are activatable by S specific target molecules (e.g., cyclin A/CDK2, cyclin B/Cdc2, SPA-), at a fourth location, nucleic acid sensor molecules activatable by G2 specific target molecules (e.g., a cell cycle biosensor).
  • pathway specific biosensors can be generated for any of apoptotic pathways, blood clotting pathways, calcium regulation pathways, cholesterol metabolism pathways, the JAK/STATS signaling pathway, MAP kinase signaling pathways, p53 pathway, PI 3 kinase pathway, ras activation pathways, SIP signaling pathways, SHC signaling pathways, TGF-13 signaling pathways, T-cell receptor complex, and MHC-I pathways, using exemplary target molecules listed above, or other target molecule components of the respective pathways.
  • pathways whose components have been characterized and that target molecules within these pathways are also encompassed within the scope of the present invention (e.g., including, but not limited to, phosphatase pathways, transcription factor pathways, hormone dependent pathways, as well as intermediary metabolism pathways, and developmental pathways).
  • additional pathways can be identified using the nucleic acid based biosensor profiling techniques discussed above (e.g., identifying pathway molecules involved in the functioning of a wild type or diseased organ system, such as the cardiovascular system, central nervous system, digestive system, reproductive system, pulmonary system, skin system, and the like), and these also are encompassed within the scope of the invention.
  • pathway specific molecules can be identified by other techniques known in the art (see, e.g., U.S. Patent Number 6,087,477, U.S. Patent Number 6,054,558, U.S. Patent Number 6,048,709, and U.S. Patent Number 6,046, 165) and used to engineer additional pathway target activatable nucleic acid sensor molecules. Because there is a finite number of pathway target molecules in each pathway (constrained by the absolute number of gene products which have been identified) (see, e.g., Drews, Science 287: 1960- 1964), it is feasible using the target activatable nucleic acid sensor molecules to generate biosensors representative of an entire pathway.
  • sets of profiling biosensors are used to monitor the expression/activity of target molecules representing complex systems.
  • target molecules representing complex systems.
  • an array representative of a plurality of systems in the human body is used in methods to assess the effects of drug compounds on multiple systems in the body.
  • the profiling nucleic acid sensor molecules according to the invention can be used in every step of a drug optimization process, as shown in Figure 8, and are suitable reagents for use in conventional high throughput screening systems making them extremely adaptable for use alone, or in conjunction with, other drug development assays.
  • profiling nucleic acid sensor molecules can be used to identify signature target molecules which are correlatable to particular traits, such as disease.
  • Signature target molecules are drug targets whose levels, structure, and/or activity can be used to evaluate the efficacy of compounds.
  • a large number of signature drug targets, both characterized and uncharacterized, can be identified simultaneously using a single profiling biosensor according to one embodiment.
  • a profiling biosensor recognizes and is independently activated by about 1-5,000 molecules.
  • a profiling biosensor recognizes and is independently activated by about 500- 10,000 molecules, and in another embodiment, by greater than 10,000 molecules.
  • the drug targets identified in step 1 are evaluated in high throughput screening assays, using either solution-based biosensors or substrate-based biosensors, to characterize the biological activity of a drug target.
  • nucleic acid sensor molecules are used to assess levels of substrate, product and intermediates produced by an enzyme in a wild type vs. a disease state, to identify other components of a pathway that would be affected by a drug acting on that target (i.e., secondary drug targets).
  • the levels, structure, and/or activity of all of the modified forms of a drug target, or the active and inactive forms of a drug target is determined in a wild type vs.
  • a disease state to further develop a diagnostic profile of a diagnostic pathway target molecule and to evaluate changes of that profile in the presence of a drug.
  • the same type of profiling biosensor used to identify a diagnostic profile is contacted with samples from patients exposed to a compound.
  • a compound-treated sample which produces substantially similar levels, structure, and/or activity of target and secondary drug targets in a sample from a healthy patient is used to identify a compound as a candidate drug. Because this testing is done in a high throughput format, a single dose of a candidate drug is evaluated in any given test.
  • the nucleic acid sensor molecules used in step 2 are tested in an in vitro biochemical assay to determine compound potency.
  • a preliminary dosing effect is determined to identify the IC50 of candidate drug.
  • multiple biosensors of the type used in step 2 are contacted with samples from patients exposed to different doses of the candidate drugs identified in step 2, to identify candidate drugs with the highest potency (e.g., requiring the least amount of drug to generate a wild type profile or an effective drug profile.
  • nucleic acid sensor molecules are used in cellular assays where the effect of adding a compound on cell physiology is known and the researcher wants to determine that the drug is in fact acting through the drug target selected in steps 1-3.
  • a candidate drug is added to a physiological system (e.g., cell(s), tissue(s), organ(s), or a patient).
  • Cells from the physiological system are lysed and the substrate or product of an enzyme reaction is monitored using the nucleic acid sensor molecule either in an ELISA format or other solid support-based format (e.g., a profiling array) or a solution phase format.
  • cell lysates are contacted with a profiling biosensor specific for a target or pathway of interest to determine the profile of target molecules in the lysed sample.
  • the profile is then compared to the wild type profile and the disease profile to determine if the drug is operating in vivo to restore a cell to its wild type state.
  • the physiological effect of a candidate drug on a physiological system is correlated with the in vivo mechanism of action of the candidate drug.
  • pathway profiling arrays comprised of nucleic acid sensor molecules affixed to a solid support are used in cellular assays to determine the selectivity of a compound for one target in a pathway relative to other candidate targets in a signal transduction pathway(s) or in another biochemical pathway(s). This data can be used to validate a drug lead or drug target.
  • nucleic acid sensor molecules are expressed in vivo or intracellularly using plasmids, viruses or other extra-chromosomal DNA vectors and the cellular nucleic acid sensor molecules are extracted and used to determine the activity of a drug or drug target.
  • These cellular assays can also determine the selectivity of a compound for one target in a pathway relative to other candidate targets in a signal transduction pathway(s) or in another biochemical pathway(s). This data can be used to validate a drug lead or drug target.
  • Step 5 drug-lead potency, specificity, and/or in vivo activity is optimized by an iterative repetition of any or all of steps 1-4. In one embodiment, steps 1-4 are repeated until the desired potency, selectivity and in vivo mechanism of action of a candidate drug is obtained. Potency can range from picomolar affinity to nanomolar affinity as measured by in vitro IC50 values. The desired selectivity of a drug candidate for its target can vary from 2 to a million-fold, and can be obtained by measuring the potency (IC50) of a drug lead toward the drug target, versus the drug's potency (IC50) values against other pertinent targets (target pertinence is determined by the requirements of the biological system under investigation).
  • IC50 potency
  • IC50 drug's potency
  • a drug lead is deemed optimal when the parameters of potency, selectivity and cellular action are optimized with respect to each other.
  • known drug leads from Steps 1-4 are found to be specific for targets that were not known to the researcher in step 2. This is also termed target discovery and validation, and occurs when steps 1-4 are repeated in an iterative fashion of any or all steps and the drug target is identified by the profiling array to, in fact, exist in an alternative signal transduction pathway, or to be a novel protein or enzyme in the pathway originally under investigation.
  • MPP arrays can identify the site of action of a drug lead, and can determine the relative selectivity of a drug for one drug target of a drug target pathway.
  • target cells e.g., tissue(s)
  • the lysate is contacted with nucleic acid sensor molecules either in a solid phase assay, a solution phase assay, or in a pathway profiling biosensor array format to assess the in vivo biological activity of a candidate drug identified by any of the previous steps or by some other method, on a target or pathway.
  • nucleic acid sensor molecules either in a solid phase assay, a solution phase assay, or in a pathway profiling biosensor array format to assess the in vivo biological activity of a candidate drug identified by any of the previous steps or by some other method, on a target or pathway.
  • Step 7 Optimization of the Drug Lead
  • drug-lead potency, specificity, and/or in vivo activity are optimized by an iterative repetition of any or all of steps 1-6.
  • steps 1-5 are repeated until the desired potency, selectivity and in vivo mechanism of action of a candidate drug are obtained.
  • the nucleic acid sensor molecules are used in pharmaco-kinetic studies, where the effect of a drug on the physiology of a cell, group of cells, tissue(s), organ(s), or animal model is assessed by obtaining blood, plasma, tissue, or a cell, and contacting this material with nucleic acid sensor molecules either in a solid phase (e.g., ELISA), solution or array format to assess the in vivo pharmacological or toxicological activity of a compound.
  • the nucleic acid sensor molecules used are developed against the candidate drug itself, its metabolic products, and/or the metabolic products of proteins and small ligands involved in a xenobiotic or toxicological response to drug treatment.
  • nucleic acid sensor molecules are employed to follow the fate of a drug or its metabolic by-products.
  • nucleic acid sensor molecules are generated to the drug and its metabolites.
  • the drug is administered to the test animal either subcutaneously, intraperitoneally or by gavage.
  • the blood plasma or disease tissue is removed and its contents are screened for the remaining drug by Liquid chromatography (LC) or LC-mass spectrometry.
  • Drug exposure is then determined as a function of time, dose and method of administration and is reported in values of half-life, bioavailability, AUC and Cmax. Metabolic products of a drug lead can be similarly followed.
  • Nucleic acid sensor molecules generated against enzymes or proteins known to those skilled in the art to be involved in drug metabolism P450 enzymes, multi-drug transporter
  • P450 enzymes, multi-drug transporter can be used to follow the effect of a drug on xenobiotic or toxicological response to drug treatment.
  • drug-lead potency, specificity, and/or in vivo activity, and pharmacokinetic, or toxological properties are optimized by an iterative repetition repetition of any or all of steps 1-7. In one embodiment, steps 1-7 are repeated until the desired potency, selectivity and in vivo activity and pharmaco-kinetic, or toxicological properties of a candidate drug are obtained.
  • nucleic acid sensor molecules are used in clinical trials to determine the fate of a drug in human or animal models, or used to follow the effect of drug treatment on a target or molecular pathway of choice, as described above.
  • the nucleic acid sensor molecules in a solid phase assay (e.g., ELISA format), a solution phase assay, or in a pathway profiling biosensor array format, are used to assess the in vivo biological activity of a drug being tested using lysed cell samples as described above.
  • the appropriate profiling biosensor is used in vivo, to monitor the effects of the compound on the patient, for example, by providing the biosensor in communication with a fiber optic probe inserted into the patient, or ex vivo, monitoring optical signals in a cell using a microscope based detection system.
  • an in vivo assay is done by introducing a nucleic acid sensor molecule which retains its catalytic activity into a physiological system (e.g., by injection at a target site in the body, through liposome carriers, and other means of administration routinely used in the art), obtaining cells from the physiological system and detecting the effect of the compound on the catalytic activity of the nucleic acid sensor molecule (e.g., by evaluating the sequence of the nucleic acid sensor molecule) as a means of determining the level, structure, or activity of a drug target, and relating the level, structure, or activity or the target molecules to the efficacy of the drug.
  • Step 11 Optimization of the Drug Lead
  • any or all of steps 1-10 are repeated to further optimize the properties of the candidate drug.
  • individuals who would be suitable for treatment with the candidate drugs identified steps 1-11 are identified using nucleic acid sensor molecules in the diagnostic assays discussed previously.
  • nucleic acid sensor molecules are used in chemical genomic assays in which a drug or plurality of drug leads, with known or unknown physiological effects, and with unknown targets, are contacted with a physiological system and the site of action of the drug or plurality of drugs is determined using a plurality of the profiling biosensors described previously. Drug optimization then occurs as in steps 1-11.
  • nucleic acid sensor molecules according to the invention can also be used to retrieve the target molecules which they specifically recognize. Additional embodiments exploiting the recognition capacity of the biosensors disclosed are contemplated and encompassed within the scope.
  • reagents are provided for generating and using nucleic acid sensor molecules.
  • a kit comprising standardized reagents for making and/or using the nucleic acid sensor molecules according to the invention.
  • the kit comprises at least a first optical nucleic acid sensor molecule whose optical properties change upon recognition of a target molecule.
  • the kit additionally comprises any of: a control target molecule, a second nucleic acid sensor molecule which recognizess a different target molecule, suitable buffers, printed instructions, and combinations thereof.
  • a nucleic acid sensor molecule is provided with reagents for attaching a label and/or quencher or with reagents for attaching charge transfer molecules to the nucleic acid sensor molecule, which can sensitize the optical properties of the nucleic acid molecule to the presence of a target molecule.
  • a composition is provided comprising a target molecule and a nucleic acid sensor molecule. The composition provides a reference against which to compare modified nucleic acid sensor molecules which recognizes to the same target, in order to select those with preferred cataytic activity or conformational change in the presence of the target.
  • sets of complexes are provided.
  • a set of pathway target molecules and nucleic acid sensor molecules are provided.
  • a set of profiling target molecules and nucleic acid sensor molecules are provided.
  • solid supports are provided for isolation of target molecules from nucleic acid sensor molecules.
  • a computer program product comprising stored data relating to optical signals generated by profiling and or pathway target molecules.
  • a means to compare this data to other optical signals is provided.
  • the memory comprises data relating to patient information or chemical structure information relating to either target molecules or nucleic acid sensor molecules.
  • the system is a robotic workstation, comprising, at least one of an: arrayer, microplate or microarray feeders, stackers, washers, and dispensers, an optical system, a carousel, a conveyer for conveying microplates or microarrays from one part of the system to another (in a vertical or horizontal direction), a shaker system or other mixing system, a temperature control system, a synthesizer, a solid phase extraction system, and sample concentrators.
  • Components of the robotic workstation can be part of a single integrated system or can be provided separately for use at any stage of the drug optimization process according to the invention.
  • the system comprises a processor connectable to the network which comprises or can access applications comprising stored data relating to profiling information obtained using nucleic acid sensor molecules according to the invention, and/or statistical applications, applications for performing structure/activity analysis of target molecules and nucleic acid sensor molecules, applications for performing nucleic acid sequence alignment and simultaneous structure superposition of proteins (e.g., MOE-Align'TM), applications for predicting binding conformations of molecules to receptor structures, and applications for controlling the processing functions of the robotic workstations.
  • a processor connectable to the network which comprises or can access applications comprising stored data relating to profiling information obtained using nucleic acid sensor molecules according to the invention, and/or statistical applications, applications for performing structure/activity analysis of target molecules and nucleic acid sensor molecules, applications for performing nucleic acid sequence alignment and simultaneous structure superposition of proteins (e.g., MOE-Align'TM), applications for predicting binding conformations of molecules to receptor structures, and applications for controlling the processing functions of the robotic workstations.
  • the invention is further illustrated in the following non
  • Target modulated nucleic acid sensor molecules are isolated by in vitro selection. Pools of partially randomized ribozymes with 10 15 -10 17 unique sequences serve as the starting point for in vitro selection. As with the engineering approach, both the LI ligase and hammerhead ribozyme are used as platforms for the selection of allosterically-controlled molecules. Selections are designed to yield ribozymes that specifically respond to any target. Nucleic acid sensor molecules with cross-specificity (i.e. modulated by alternate target molecules, or by alternate ligand-bound states, or by alternate post-translationally modified forms of protein or peptides) are selected against by including the undesired form of the target in an initial negative selection step. The specific sequence of operations comprising the selection experiment is outlined in Figure IA and IB for ligase and hammerhead-based selections, respectively
  • the starting library of DNA sequences is generated by automated chemical synthesis on a DNA synthesizer. This library of sequences is transcribed in vitro into RNA using T7 RNA polymerase, purified, and captured onto beads using an oligonucleotide tag complementary to the 3 '-end.
  • RNA library In the absence of the desired modulator (target), the RNA library are incubated together with the undesired form of the modulator and an arbitrary sequence oligonucleotide substrate (substrate 1). (During this incubation, non-modulated ribozymes undergo ligation ( Figure IA) or cleavage ( Figure IB).
  • Ligases active only in the presence of the desired modulator are isolated using streptavidin capture and selective PCR amplification (relying on sequence differences between substrates 1 and 2 to distinguish allosteric and non-allosteric activities). Hammerheads active only in presence of the desired modulator are isolated by gel electrophoresis. 5. Purification. PCR amplified DNA are purified and transcribed to yield an enriched pool for subsequent reselection.
  • NASMs have been selected as described above, they are characterized as follows:
  • Nucleic acid sensor molecules which are derived from in vitro selection are tested as target modulated biosensors.
  • the pool of NASMs is cloned into various plasmids that contain a T7 promoter transformed into E. coli.
  • Individual NASM encoded DNA clones are isolated, linearized and the NASM is transcribed in vitro to generate NASM RNA.
  • the NASM RNAs are then tested in target modulation assays which determine the rate or extent of ribozyme modulation.
  • target modulation assays which determine the rate or extent of ribozyme modulation.
  • the extent of target dependent and independent reaction is determined by quantifying the extent of endonucleolytic cleavage of an oligonucleotide substrate.
  • the extent of reaction can be followed by electrophoresing the reaction products on a denaturing PAGE gel, and subsequently analyzed by standard radiometric methods.
  • the extent of target dependent and independent reaction is determined by quantifying the extent of ligation of an oligonucleotide substrate, resulting in an increase in NASM molecular weight, as determined in denaturing PAGE gel electrophoresis.
  • NASM sequences are then further modified to render (the NASM sequences) them suitable for the optical detection applications that are described in detail below.
  • these NASMs are used as fluorescent biosensors affixed to solid supports, as fluorescent biosensors in homogeneous FRET-based assays, and as biosensors in SPR applications.
  • Ligase derived NASM clones are further modified to render them suitable for a number of detection platforms and applications; including, but not limited to, the PCR and nucleotide amplification detection methods; fluorescent-based biosensors detectable in solution and chip formats; and as in vivo, intracellular detection biosensors.
  • detection platforms and applications including, but not limited to, the PCR and nucleotide amplification detection methods; fluorescent-based biosensors detectable in solution and chip formats; and as in vivo, intracellular detection biosensors.
  • the various detection applications of hammerhead ligase and intron-based NASMs are described in detail below.
  • the following protocol describes a method for preparing an array of immobilized effector oligonucleotides with terminal amine groups attached to a solid substrate derivatized with aldehyde groups.
  • the resulting array can then be used to spatially address (i.e., the sequence of nucleotides for each effector oligonucleotide can be synthesized as a cognate to the effector oligonucleotide binding domain of a nucleic acid sensor molecule specific for a particular target molecule) and immobilize the nucleic acid sensor molecules prior to their use in a solid-phase assay (see, e.g., Zammatteo et al., 2000):
  • the nucleic acid sensor molecules can be, e.g., those which possess either ligating or cleaving activity in the presence of a target molecule. (See, e.g., Figures 2A and B for the ligater, Figure 5 for the cleaver).
  • the nucleic acid sensor molecules are bound to a solid substrate directly via their 3' termini.
  • the attachment is accomplished by oxidation (using, e.g., Na periodate) of the 3' vicinal diol of the nucleic acid sensor molecule to an aldehyde group.
  • This aldehyde group will react with a hydrazide group to form a hydrazone bond.
  • the hydrazone bond is quite stable to hydrolysis, etc., but can be further reduced (for example, by treatment with NaBH or NaCNBH 3 ).
  • adipic acid dihydrazide (ADH, a bifunctional linker) to derivatize an aldehyde surface results in a hydrazide- derivatized surface which provides a linker of approximately 10 atoms between the substrate surface and point of biomolecular attachment (see Ruhn et al., 1994;
  • Preparation of a hydrazide-terminated surface via ADH treatment can be accomplished by treating an aldehyde-derivatized substrate according to the following protocol:
  • a nucleic acid sensor molecule which is modulated by the estrogen receptor (ER) ligand binding domain (LBD) is obtained by in vitro selection methods to identify candidate nucleic acid sensor molecules that are modulated by an estrogen receptor LBD.
  • the full length gene for the estrogen receptor is known.
  • One source of the full- length estrogen receptor clone is Ace. No. M12674 (see also Greene et al., Science 231 :1150-54, 1986).
  • the clone includes a 2092 nucleotide mRNA with the sequence presented in Table 3 below:
  • Table 3 1 gaattccaaa attgtgatgt ttcttgtatt tttgatgaag gagaaatact gtaatgatca
  • polypeptide encodes a polypeptide with the amino acid sequence presented in Table 4 below:
  • nucleic acid sensor molecules which are activated by ER-LBD not bound to ligand A library of up to IO 17 variants of in vitro synthesized (1 ⁇ M) nucleic acid sensor molecules is allowed to react with purified apo-ER-LBD at a final concentration of luM. Selection of catalytic nucleic acid sensor molecules and optionally, generation of an optical NASM, is carried out by procedures outlined in prior examples and elsewhere herein.
  • nucleic acid sensor molecules which are modulated by the ER-LBD- Estradiol complex Stable complexes of ER-LBD and estradiol ligand are formed with from 1-10 equivalents of ligand. A library of up to IO 17 variants of in vitro synthesized ribozymes is then allowed to react with purified ER-LBD-Estradiol at a final complex concentration of luM. Selection of allosterically activated ribozymes is carried out by procedures outlined the detailed description.
  • nucleic acid sensor molecules which are modulated by the ER-LBD-Tamoxifen complex stable complexes of ER-LBD and tamoxifen ligand are fo ⁇ ned with from 1-10 equivalents of ligand.
  • a library of up to IO 17 variants of in vitro synthesized ribozymes (1 ⁇ M containing a plurality of potential target modulation domains and linker domains coupled to the catalytic domain of the ribozyme, is then allowed to react with purified ER-LBD-Tamoxifen at a final complex concentration of luM.
  • Selection of catalytic nucleic acid sensor molecules target modulated ribozymes is carried out by procedures outlined in prior examples.
  • NHR nuclear hormone receptor
  • N-terminally GST-tagged or N-/C -terminally His-tagged nuclear hormone receptor ligand binding domains defined on the basis of structural homology are cloned and expressed in BL21 (DE3)-pLysS E. coli cells, or are cloned and expressed in standard baculovirus expression systems.
  • Human NHR LBDs (homologous to ER-alpha residues including the region aa 297- 554) are purified from GSH-sepharose or nickel affinity columns by published procedures available from the manufacturers. LBDs are produced in a either a parallel or serial fashion and the purified proteins are stored in buffer containing 50 mM TrisHCl, 1 mM EDTA, ImM DTT and 50-250 NaCl/SCN salt, pH 7 to pH 8.5, 10% glycerol or other stabilizing agent. Protein sequence and MW is verified by electrospray LC-MS mass spectrometry.
  • nucleic acid sensor molecules which are modulated by NHR-LBD not bound to ligand A library of up to IO 17 variants of in vitro synthesized ribozymes (1 ⁇ M) containing a plurality of potential target modulation domains and linker domains coupled to the catalytic domain of the ribozyme, is allowed to react with purified apo-NHR- LBDs at a final concentration of 1 uM LBD. Selection of catalytic nucleic acid sensor molecules is carried out by procedures outlined in the Detailed Description. Selections are carried out in parallel fashion. Selections can also be carried out in mixed pools of anywhere from 5-10 NHR-LBDs.
  • RNA pools may separated into aliquots which may then be used to carry out in vitro selection against single NHR-LBD proteins to yield unique nucleic acid sensor molecules selective for multiple NHR-LBDs.
  • Stable complexes of each NHR-LBD are formed with from 1-10 equivalents of ligand.
  • a library of up to IO 17 variants of in vitro synthesized ribozymes (1 ⁇ M) containing a plurality of potential target modulation domains and linker domains coupled to the catalytic domain of the ribozyme is then allowed to react with purified NHR-LBD-Ligand complexes at a final complex concentration of 1 uM.
  • Selection of catalytic nucleic acid sensor molecules is carried out by procedures outlined in the Detailed Description and Example 1 below. Selections are carried out in parallel fashion. Selections can also be carried out in mixed pools of anywhere from 5-10 NHR-LBD-Ligand complexes.
  • RNA pools may separated into aliquots which may then be used to carry out in vitro selection against single NHR-LBD- Ligand complexes to yield unique nucleic acid sensor molecules selective for all NHR- LBDs, bound to their ligands.
  • the invention provides an in vitro selection protocol against purified LBDs, bound to their ligands or not, for each known NHR.
  • In vitro selections can be carried out with less than 1 mg of the purified forms of the LBDs.
  • the selection of nucleic acid sensor molecules can be done in vitro with mixed pools of LBD and subsequently deconvoluted after selection is complete.
  • the final selection can be carried out with fractionally purified extracts containing a slight excess of recombinant LBD.
  • the LBD is expressed in E. coli, or insect cell lines or mammalian cell lines.
  • the selection is carried out in cell free lysates in which the LBD is expressed in an in vitro transcription-translation procedure such as is described in the literature or can be purchased using common reagents from Roche or Promega.
  • the fractionated or purified LBDs are combined with known ligands, (as described above) agonist, antagonists or partial agonist/antagonists to form stable complexes, and these complexes are then used for in vitro selection of nucleic acid sensor molecules.
  • known ligands, (as described above) agonist, antagonists or partial agonist/antagonists to form stable complexes, and these complexes are then used for in vitro selection of nucleic acid sensor molecules.
  • a signal Upon interaction of the nucleic acid sensor molecule with the NHR-LBD, a signal will be generated detectable to an external monitoring device. In this manner, the activation state of any or all NHRs can be monitored in vivo or in vitro as will be described in detail in subsequent examples.
  • Example 4 Selection for a nucleic acid sensor molecule selective for the Beta-2 adrenergic receptor.
  • the full-length gene for the Beta-2 adrenergic receptor is described (Emorine et al, Proc. Natl. Acad. Sci. USA 84:6995-99, 1987) and is available at Ace. No. AAA88017.
  • the nucleic acid sequence is set forth in Table 6 below:
  • amino acid sequence of the polypeptide encoded by the nucleic acid sequence is set forth in Table 7 below:
  • Table 7 1 mgqpgngsaf llapngshap dhdvtqqrde vwwgmgivm slivlaivfg nvlvitaiak 61 ferlqtvtny fitslacadl vmglawpfg aahilmkmwt fgnfwcefwt sidvlcvtas 121 ietlcviavd ryfaitspfk yqslltknka rviilmvwiv sgltsflpiq mhwyrathqe 181 aincyanetc cdfftnqaya iassivsfyv plvimvfvys rvfqeakrql qkidksegrf 241 hvqnlsqveq dgrtghglrr sskfclkehk alktlgiim
  • Beta-2 adrenergic receptor or cytoplasmic loop II or III or helix VII is cloned, expressed and purified from E. coli or baculovirus infected cells (Hampe, et al., J Biotechnol 77:219-234 (2000)) according to published procedures, and incorporated into detergent micelles to simulate the cellular milieu (Min, et al., J Biol Chem 268:9400-9404 (1993)).
  • a library of up to IO 17 variants of in vitro synthesized ribozymes (1 ⁇ M) containing a plurality of potential target modulation domains and linker domains coupled to the catalytic domain of the ribozyme is allowed to react with purified Beta-2 adrenergic receptor at a final concentration of 1 uM.
  • Selection of catalytic nucleic acid sensor molecules is carried out by procedures outlined in Example 1. Selection of nucleic acid sensor molecules which are activated by the butoxamine-
  • Beta-2 adrenergic complex 1 :1 complexes of butoxamine and purified Beta-2 adrenergic receptor are formed and selection of catalytic nucleic acid sensor molecules is carried out by procedures outlined in Examplel.
  • nucleic acid sensor molecules which are activated by the isoproterenol- Beta-2 adrenergic complex is accomplished as follows. 1:1 complexes of isoproterenol and purified Beta-2 adrenergic receptor are formed and selection of catalytic nucleic acid sensor molecules is carried out by procedures outlined in Example 1 and the Detailed Description.
  • Example 5 Selection for a library of nucleic acid sensor molecules which signal the presence of all known GPCRs
  • GPCR G-protein coupled receptors
  • All 400 plus GPCRs are produced in either a serial or parallel fashion and the purified proteins or peptides stored in buffer containing 50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, and 50-250 mM NaCl/SCN salt, pH 7 to pH 8.5, 10% glycerol or other stabilizing agent.
  • Table 8 ref NP_ _006134 .l
  • 46228 [Homo sapiens] >gi
  • ref XP_ _001777 lj orexin receptor 1 [Homo sapiens] ref XP_ _011871 3 j neuropeptide FF 1; RFamide-related peptide.
  • ref NP_ _001516 lj orexin receptor 1 [Homo sapiens] ref NP_ _001041 1 j somatostatin receptor 2 [Homo sapiens] >gi .
  • ref NP_ _001040 1 j somatostatin receptor 1 [Homo sapiens] >gi .
  • ref NP_ _006670 1 j putative opioid receptor, neuromedin K (ne. ref NP_ _055442 1 j G protein-coupled receptor 57 [Homo sapiens] ref NP_ _000698 lj arginine vasopressin receptor IB; arginine.
  • ref XP_ _040306 lj similar to SOMATOSTATIN RECEPTOR TYPE 2 S. ref NP_ _056542 lj tachykinin receptor 1, isoform short; Tach.
  • ref NP ⁇ _001042 lj somatostatin receptor 3 [Homo sapiens] >gi .
  • ref NP_ _000722 lj cholecystokinin B receptor [Homo sapiens] ref NP_ _000789 ⁇
  • ref NP_ 004215 lj G protein-coupled receptor 50 [Homo sapiens] ref XP_ _010228 2 j G protein- coupled receptor 50 [Homo sapiens] ref NP_ 000907 lj oxytocin receptor [Homo sapiens] ref XP_ 052179 lj oxytocin receptor [Homo sapiens] >gi
  • G protein-coupled receptor 57 [Homo sapiens] melatonin receptor IA; melatonin receptor .. G protein-coupled receptor sit; melanin-co.. opioid receptor, kappa 1; Opiate receptor,., neuropeptide Y receptor Yl; Neuropeptide Y.. similar to opioid receptor, kappa 1; Opiat.. adrenergic, alpha-lA-, receptor [Homo sapi ..
  • cholinergic receptor muscarinic 4; muscar.. 44527 [Homo sapiens] dopamine receptor D3 [Homo sapiens] dopamine receptor D3 [Homo sapiens] G protein-coupled receptor 14 [Homo sapiens] cholinergic receptor, muscarinic 3; muscar.. adrenergic, alpha-ID-, receptor; adrenergi.. G protein-coupled receptor 17 [Homo sapien.. similar to purinergic receptor (family A g.. similar to G protein-coupled receptor 17 (.. angiotensin receptor-like 1 [Homo sapiens] .. adrenergic, beta-3-, receptor [Homo sapien..
  • chemokine (C-C motif) receptor 1 macropha.. opioid receptor, delta 1 [Homo sapiens] purinergic receptor (family A group 5) [Ho., cholinergic receptor, muscarinic 4 [Homo s.. similar to MUSCARINIC ACETYLCHOLINE RECEPT... cholinergic receptor, muscarinic 1; muscar.. opiate receptor-like 1; opiod receptor-lik .. cysteinyl leukotriene receptor 1 [Homo sap .. chemokine (C-C motif) receptor 3 [Homo sap., rhodopsin; rhodopsin (retinitis pigmentosa..
  • G protein coupled receptor [Homo .. adenosine A2a receptor [Homo sapiens] adrenergic, alpha-ID-, receptor [Homo sapi.. gonadotropin-releasing hormone receptor; g.. adenosine A2a receptor; adenosine A2 recep.. G protein-coupled receptor 45 [Homo sapiens] putative neurotransmitter receptor [Homo s .. 5-hydroxytryptamine (serotonin) receptor 1.. opioid receptor, delta 1 [Homo sapiens] >g.. melatonin receptor IB; melatonin receptor ..
  • interleukin 8 receptor beta [Homo sapiens] neuromedin U receptor 2 [Homo sapiens] G protein-coupled receptor 45 [Homo sapiens] interleukin 8 receptor, alpha; chemokine (.. putative neurotransmitter receptor [Homo s .. cholinergic receptor, muscarinic 5; muscar.. leukotriene b4 receptor (chemokine recepto..
  • dopamine receptor D4 [Homo sapiens] dopamine receptor D4 [Homo sapiens] G protein-coupled receptor 52 [Homo sapiens] G protein-coupled receptor 8 [Homo sapiens] purinergic receptor (family A group 5) [Ho., neuropeptide Y receptor Y5 [Homo sapiens] neurotensin receptor 1 [Homo sapiens] 5-hydroxytryptamine (serotonin) receptor 1..
  • neurotensin receptor 1 [Homo sapiens] G protein-coupled receptor 8 [Homo sapiens] 5-hydroxytryptamine (serotonin) receptor 1. Burkitt lymphoma receptor 1, isoform 1; C- .
  • G protein-coupled receptor 52 [Homo sapiens histamine receptor HI; histamine receptor,, chemokine (C-C motif) receptor 2; chemokin. Burkitt lymphoma receptor 1, isoform 2; C- . retinal pigment epithelium-derived rhodops.
  • G-protein coupled receptor [Homo sapiens] . formyl peptide receptor-like 2 [Homo sapie. 5-hydroxytryptamine (serotonin) receptor 5.
  • G protein-coupled receptor 7 [Homo sapiens]
  • G protein-coupled receptor 91 [Homo sapien. neuropeptide Y receptor Y6 (pseudogene) [H.
  • galanin receptor 1 [Homo sapiens] >gi
  • G protein-coupled receptor 15 [Homo sapien. angiotensin receptor 2 [Homo sapiens] >gi
  • G protein-coupled receptor 24 [Homo sapien. formyl peptide receptor-like 2 [Homo sapie.
  • G protein-coupled receptor 7 [Homo sapiens] similar to somatostatin receptor-like prot. chemokine (C-X-C motif) , receptor 4 (fusin. adrenergic, alpha-2B-, receptor [Homo sapi. similar to C-X-C CHEMOKINE RECEPTOR TYPE 4. formyl peptide receptor-like 1; lipoxin A4.
  • G protein-coupled receptor 66 [Homo sapien. 5-hydroxytryptamine (serotonin) receptor 1.
  • 41064 [Homo sapiens] chemokine (C-C motif) receptor 4; chemokin. chemokine (C-C motif) receptor 2; chemokin.
  • opiate receptor-like 1 [Homo sapiens] >gi
  • G protein-coupled receptor 21 [Homo sapien. 34426 [Homo sapiens] chemokine-like receptor 1 [Homo sapiens] >.
  • chemokine-like receptor 1 [Homo sapiens] ref NP__000855 l
  • ref NP_ _000045 lj arginine vasopressin receptor 2 [Homo sapi.
  • ref XP_ _048964 1 j similar to PROBABLE G PROTEIN-COUPLED RECE .
  • ref XP_ _011880 lj similar to pancreatic polypeptide receptor, ref NP_ _000676 lj angiotensin receptor 1; angiotensin recept.
  • ref XP_ _002705 3 j G protein-coupled receptor 17 [Homo sapiens] ref NP_ _000677 lj angiotensin receptor 2 [Homo sapiens] ref ⁇ p ⁇ _004279 lj chemokine (C-C motif) receptor 6 [Homo sap., ref NP_ _000504 lj opsin 1 (cone pigments), medium-wave-sensi .. ref XP_ _033840 lj similar to chemokine (C-C motif) receptor .. ref NP_ _002555 ⁇
  • ref NP_ _005274 1 j G protein-coupled receptor 5 [Homo sapiens., ref NP_ _061842 lj super conserved receptor expressed in brai .. ref NP_ _005291 lj G protein-coupled receptor 34 [Homo sapien.. ref NP_ 037477 lj G protein-coupled receptor [Homo sapiens] .. ref XP_ _003126 lj chemokine binding protein 2 [Homo sapiens] ..
  • ref XP_ _007392 1 j G protein-coupled receptor 65 [Homo sapiens] ref NP_ _005284 lj G protein-coupled receptor 20 [Homo sapiens] ref NP_ _005287 lj G protein-coupled receptor 23 [Homo sapien.. ref NP_ _0Q9195 ⁇
  • ref NP_ _005273 lj G protein-coupled receptor 4 [Homo sapiens., ref NP_ _000668 lj adenosine A3 receptor [Homo sapiens] >gi
  • endothelin receptor type B isoform 1 [Horn.. ref NP_ _003982 lj endothelin receptor type B isoform 2 [Homo., ref xp . _007276 2 j bradykinin receptor B2 [Homo sapiens] >gi
  • ref XP__009029 4 purinergic receptor P2Y, G-protein coupled, ref NP_ _036284 ⁇
  • ref NP_ 055694 ⁇
  • ref NP_ _055314 lj putative purinergic receptor [Homo sapiens, ref NP_ 036509 1 j olfactory receptor, family 7, subfamily C, . ref NP_ _036507 1 j olfactory receptor, family 52, subfamily A.
  • ref NP ⁇ _061844 ⁇
  • ref NP_ 063941 lj melanocortin 3 receptor [Homo sapiens] ref NP ⁇ _009091 1 j olfactory receptor, family 2, subfamily H, . ref XP_ _008678 2 j olfactory receptor, family 1, subfamily D, . ref NP_ _003543 1 j olfactory receptor, family 1, subfamily D, . ref XP_ 011731 3 j similar to adrenergic, beta-3-, receptor (.
  • melanocortin 3 receptor [Homo sapiens] ref NP_ 076403 lj G protein-coupled receptor 86 [Homo sapiens] ref XP_ 042200 lj G protein-coupled receptor 86 [Homo sapiens] ref NP_ _004711 21 endothelial differentiation, lysophosphati . ref NP_ _037440 1 j platelet activating receptor homolog [Homo, ref NP_ _005217 1 j endothelial differentiation, sphingolipid . ref NP ⁇ _005295 ⁇
  • nucleic acid sensor molecules which are activated by GPCRs not bound to ligand A library of up to IO 17 variants of in vitro synthesized ribozymes is allowed to react with purified GPCRs at a final concentration of luM GPCR. Selection of catalytic nucleic acid sensor molecules is carried out by procedures outlined in prior examples. Selections are carried out in parallel fashion. Selections can also be carried out in mixed pools of anywhere from 5-10 GPCRs. In the final rounds of nucleic acid sensor molecule selection, the RNA pools may separated into aliquots which may then be used to carry out in vitro selection against single GPCR proteins to yield unique nucleic acid sensor molecules selective for all 400 plus GPCRs.
  • RNA pools may separated into aliquots which may then be used to carry out in vitro selection against single GPCR-ligand complexes to yield unique nucleic acid sensor molecules selective for all GPCR-ligand complexes.
  • nucleic acid sensor molecules that specifically recognize conformational isoforms of GPCRs that are revealed upon ligand binding can also be selected for using the methods described herein.
  • GPCRs consist of three domains: an extracellular N- terminus, a central domain of seven trans-membrane helices, and a cytoplasmic C-terminus.
  • Activation of GPCRs is induced by ligand binding, which causes a conformational change in the receptor transmitting a signal across the plasma membrane to intracellular members of a signaling pathway. This method provides for generation of unique biosensors for each GPCR.
  • a library of up to IO 17 variants of in vitro synthesized ribozymes is allows to react with peptide fragments of the GPCRs comprising regions of the GPCR that are exposed upon activation.
  • Nucleic acid sensor molecules which recognize these domains are then capable of recognizing them within the context of the full length protein and hence recognize the activated state of the GPCR.
  • Examples of the use of peptide fragments to generate nucleic acid sensor molecules which recognize the full length protein are known in the art and are incorporated herein. See, for example, data on nucleic acid sensor molecule selection and recognition of HIV rev peptide and full length protein [Michael Robertson, 2001, University of Texas, Austin, Ph.D. Dissertation].
  • unique peptide sequences are recognized both as free peptides and in the context of the full protein.
  • Nucleic acid sensor molecules specific for GPCRs are generated by in vitro selection for recognition of peptide fragments of the GPCRs comprising regions of the GPCR that are exposed to the inside face of the plasma membrane when ligand binds to the GPCR.
  • Exemplary suitable GPCR peptide fragments are presented in Table 9. Nucleic acid sensor molecules which recognize these peptides are then capable of recognizing them within the context of the full length protein and hence recognize the activated state of the GPCR.
  • Nucleic acid sensor molecules are generated to be specific to various subclasses of PDEs are used for understanding the role of PDE subclasses in the molecular pathology of disease, and as PDE target validation.
  • nucleic acid sensor molecules which are modulated by each of four PDE4 subtypes have specific utility in understanding the role of PDE4 in human disease.
  • the four subclasses of PDE4 are differentially localized between cell type and also the PDE4 isozymes differ with respect to their intracellular localization. This differential localization, together with the transcriptional regulation and post-translational modification, controls the cAMP level in cells in response to the cells' environment (Muller, Engels et al. 1996).
  • the cDNAs for four PDE4 subtypes are cloned from human blood leukocyte cDNA library as described (Wang, Myers et al. 1997). Each subclass of PDE4 is expressed as recombinant protein fused with a His-tag in E. coli or SF9 insect cells (Richter, Hermsdorf et al. 2000) (Wang, Myers et al. 1997). The expressed proteins are purified through Ni ++ columns according to established procedures. Catalytic nucleic acid sensor molecules modulated by the four subclasses of PDE4 are then selected as described above. The nucleic acid sensor molecules are tested for their subclass specificity, by determining the switch factor.
  • Tissue samples from different organs can be prepared, and the cell extract can be tested against a panel of PDE4 subclass-specific nucleic acid sensor molecules to determine the protein level of each PDE in the organ.
  • PDE4 subclass-specific nucleic acid sensor molecules to determine the protein level of each PDE in the organ.
  • PDE1-1 1 different classes of PDE (PDE1-1 1) are expressed in a tissue-specific manner and play different physiological roles (Conti 2000), and the subcellular localization of PDE regulates their activity. Accordingly, the nucleic acid sensor molecule can be used to determine the subcellular localization of each PDE from fractionated cell extracts (Bolger, Amsterdam et al. 1997), or in situ hybridization technique (Sirinarumitr, Paul et al. 1997).
  • nucleotide sequences of a cAMP-dependent PDE nucleic acid sensor molecule and cGMP-dependent nucleic acid sensor molecule are presented in Table 10. Allosteric domains are shown in bold font and the cleavage site nucleotide is underlined.
  • CGMP modulated NASMs that are configured for homogeneous, solution based fluorescence assays (FRET) are shown the Figure 62. Multiplexed camp and cGMP-modulated FRET- sensor NASM-based assays are shown in Figure 65B.
  • the optical NASMs modulated by cAMP and cGMP are used in PDE assays as described in detail below.
  • cAMP and cGMP-dependent nucleic acid sensor molecules were added to a solution containing various amounts of PDE and the corresponding cyclic nucleotide
  • cAMP or cGMP cyclic nucleotide-dependent nucleic acid sensor molecules
  • Assay conditions 10 mM Tris, 20 mM MgCl 2; 100 ⁇ M CaCl 2 ) 200 nM cGMP-NASM; the assay is quenched with 0.1% SDS and then 200 nM cGMP- NASM is added to the mixture.
  • Remaining cGMP was determined by the amount of conversion of nucleic acid sensor molecule to product using a gel based radioactive product- release (gel-shift) assay format.
  • the nucleic acid sensor molecule is active in a variety of formats and is not inhibited by GMP produced by the components of the PDE assay.
  • the cAMP nucleic acid sensor molecule is active in a variety of assay formats and is not inhibited by GMP produced by the components of the PDE assay.
  • the conversion of cGMP to GMP is followed by the optical NASM formats described in Figures 62-72.
  • HTS High Throughput Screening
  • a cAMP-dependent nucleic acid sensor molecule is used in HTS assays for PDEs (PDE1, PDE2, PDE3, PDE4, PDE7, PDE8, PDE10, and PDE11).
  • cGMP- dependent-nucleic acid sensor molecules can be used in HTS assays for PDEs (PDE5, PDE9, PDE 10, and PDE11).
  • Representative cAMP-dependent and cGMP-dependent PDE nucleic acid sensor molecules are shown above, and in Figures 62-72 for all solution and chip-based NASM assay configurations.
  • Each class of PDE can be isolated from human tissue (Ballard, Gingell et al. 1998), or expressed as recombinant proteins in various system (e.g.
  • the nucleic acid sensor molecule monitors the PDE activity in the presence and the absence of candidate drugs.
  • PDE and its substrate i.e., cAMP and/or cGMP
  • a multiwell chamber e.g., 96, 384 well
  • the reaction is terminated by changing the buffer conditions (e.g., addition of sufficient amount of EGTA, shifting buffer pH), or by separating enzyme and substrate (e.g., filtration).
  • the nucleic acid sensor molecules are added to measure the altered concentration of the substrate, cAMP and cGMP.
  • optical nucleic acid sensor molecules can be added without terminating the PDE activity.
  • cAMP- or cGMP-dependent nucleic acid sensor molecules can also be used to characterize the IC50 of the drug in vitro.
  • a PDE assay is performed with serial dilutions of a compound of interest. Purified PDE or, alternatively, soluble extract from cells (Moreland, Goldstein et al. 1998) can be used for the assay. The assay can be performed as described herein.
  • cAMP- or cGMP-dependent nucleic acid sensor molecules are used to characterize the IC50 values of drug candidate in vitro by analyzing cAMP- or cGMP synthesized by adenyl and guanyl cyclases.
  • Adenylate and guanylate cyclase assays are set up with serial dilution of a compound of interest.
  • Membrane fractions containing adenylate and guanylate cyclases are used for the assay.
  • the assay can be setup as described in the literature using ATP or GTP as the substrate.
  • Competitive assays using PDE nucleic acid sensor molecules Nucleic acid sensor molecules are generated that interact with the active sites of
  • PDEs PDE4 proteins are obtained as described above.
  • the nucleic acid sensor molecules are selected against PDE4 with negative selection in the presence of PDE4 complexed with subnanomolar inhibitor (e.g., Rolipram).
  • subnanomolar inhibitor e.g., Rolipram
  • the nucleic acid sensor molecule is modulated by free, uncomplexed PDE4, the PDE nucleic acid sensor molecules compete for PDE binding with inhibitors.
  • the direct inhibition by the nucleic acid sensor molecules can be tested using commercially available PDE assay kits (Amersham SPA assay kit for cAMP, Molecular Devices HEEP cAMP assay kit).
  • PDE assay kits Amersham SPA assay kit for cAMP, Molecular Devices HEEP cAMP assay kit.
  • the competition is performed by monitoring the signal from the nucleic acid sensor molecules in the presence of various inhibitors.
  • Purified PDE or soluble cell extract from appropriate source e.g., Wistar rat brain (Andersson, Gemalmaz et al. 1999)
  • nucleic acid sensor molecules 100 nM
  • nucleic acid sensor molecules 100 nM
  • the changes in the initial rate of each nucleic acid sensor molecule response in the presence and the absence of the drug can be monitored in homologous system. Multiple PDEs can be tested against a same compound in the same well. This assay is expanded if desired to determine the tissue specific interaction of each class of PDE and any compounds.
  • Nucleic acid sensor molecules are used to monitor the cellular cAMP and cGMP level in response to the injection of drugs in tissue or rat cell lines. For example, strips of human corpus collasum (HCC) tissue or rat HCC cell lines (NISI and McA-RH7777 cells) can be incubated in the presence and absence of a drug against PDES (Min, Kim et al. 2000) (Arora, de Groen et al. 1996), and the cGMP specific nucleic acid sensor molecule can be used to measure the amount of cGMP in soluble extract from the tissue or cell sample as described above.
  • HCC human corpus collasum
  • NESI and McA-RH7777 cells strips of human corpus collasum (HCC) tissue or rat HCC cell lines
  • the cGMP specific nucleic acid sensor molecule can be used to measure the amount of cGMP in soluble extract from the tissue or cell sample as described above.
  • the cAMP, and cGMP-dependent nucleic acid sensor molecules are incorporated into a reporter-gene plasmid as described above.
  • This construct is introduced in cell lines by standard transfection (e.g. lipid-mediated transfection, calcium-phosphate co-precipitation, microinjection, electroporation, retroviral infection). The level of cGMP or camp in the cell is measured by the expression of the reporter gene.
  • Class specific PDE assay Nucleic acid sensor molecules are selected for the catalytic domains of each class of
  • PDE 1-11 are prepared. These nucleic acid sensor molecules are then used for target validation as described above.
  • nucleic acid sensor molecules are used in competitive inhibition assays.
  • Competitive nucleic acid sensor molecules are used in in vitro assays to screen compounds against multiple PDEs in multiplex assays, as described above.
  • Nucleic acid sensor molecules modulated by drug leads or drug compounds used in preclinical testing and clinical trial for pharmacokinetics studies are selected and identified as described in the Detailed Description.
  • a human serum sample with or without the administration of a drug or other therapeutic agent is prepared (Berzas Nevado, Rodriguez Flores et al. 2001).
  • the nucleic acid sensor molecule is added to the sample.
  • the nucleic acid sensor molecule is then used in optical or PCR based detection methods as described in later examples, thereby quantifying the drug concentration in the whole serum or extract from the serum.
  • the nucleic acid sensor molecule modulated by various drugs or leads can also be used to determine the drug distribution in an animal model system.
  • a drug can be administrated in animals (e.g., Sprague Dawley rats, New Zealand white rabbit) by IV or orally (Andersson, Gemalmaz et al. 1999) (Jeremy, Ballard et al. 1997).
  • animals e.g., Sprague Dawley rats, New Zealand white rabbit
  • Oralsson Gemalmaz et al. 1999
  • Jeremy, Ballard et al. 1997 At various time intervals after drug administration, the animal is sacrificed.
  • Various organs are tested for the drug distribution by in situ hybridization using the drug-dependent nucleic acid sensor molecule.
  • each organs/serum is prepared for pharmacokinetic studies.
  • Example 8 Cell-permeability studies using nucleic acid sensor molecules Nucleic acid sensor molecules against a test compound are used to test cell permeability of the compound. These nucleic acid sensor molecules can be incorporated into a reporter gene construct, if desired, to make a drug-sensitive reporter gene system as described above. This construct is introduced in established cell lines (e.g. HELA cells, 293 cell, CHO cell). The cells are cultured in various concentrations of drug in media, and the expression of the reporter gene is monitored.
  • established cell lines e.g. HELA cells, 293 cell, CHO cell. The cells are cultured in various concentrations of drug in media, and the expression of the reporter gene is monitored.
  • Lysis Buffer 500 mM KC1, 20 mM Tris-Cl, pH 8.0, 10% glycerol, 0.5%) NP-40, supplemented with 1 Complete EDTA-Free Protease Inhibitor tablet (Roche) per 50 ml) per liter of culture.
  • Cells were frozen in liquid nitrogen and stored at -80 °C. Lysis and clarification was accomplished by rapid thawing in a 40 °C bath for 10 minutes, incubation at 4 °C for thirty minutes, and centrifugation for 60 minutes at 100,000 x g.
  • MCAC metal chelate affinity chromatography
  • ERK For purification of ERK, cells were grown to an OD600 of 0.7 at 37 °C and induced with 1 mM IPTG for 2 hours at 37 °C.
  • the MCAC fraction was diluted tenfold with Buffer B (20 mM HEPES 8.0, 1 mM DTT, 1 mM EDTA, 10% glycerol) and loaded onto a 5 ml HiTrap Q column (Amersham Biosciences) previously equilibrated with Buffer B plus 50 mM KC1.
  • Buffer B (20 mM HEPES 8.0, 1 mM DTT, 1 mM EDTA, 10% glycerol)
  • the column was washed with 5 column volumes Buffer C, 5 column volumes Buffer C plus 50 mM NaCl, eluted with a 7 column volume gradient to Buffer C plus 300 mM NaCl, and finished with 5 column volumes of Buffer C plus 300 mM NaCl.
  • the SP column was run at 0.5 ml/min collecting 0.5 ml fractions.
  • Ion-exchange chromatograpy conditions for p38gamma, h-Ras, and RhoA were identical to ERK, except KC1 was substituted for NaCl in Buffer D additions.
  • KC1 was substituted for NaCl in Buffer D additions.
  • For purification of p38gamma cultures were grown to an OD600 of 0.8 and induced with 0.5 mM IPTG for 3 hours at 30 °C.
  • RhoA and h-Ras cells were grown at 37 °C to an 0D600 of 0.8 at 37 "C, and induced with 0.5 mM IPTG for 3 hours.
  • MCAC fractions were diluted two fold with ddH 2 0, and dialyzed into Buffer D (20 mM HEPES 7.4, 1 mM DTT, 1 mM EDTA, 10% glycerol) plus 50 mM NaCl. Dialysate was applied to a 1 ml HiTrap Q column (Amersham Biosciences), washed with 5 column volumes Buffer D plus 50 mM NaCl, eluted with 8 column volumes to Buffer D plus 500 mM NaCl, and flushed with 3 column volumes Buffer D plus 500 M NaCl. Column flow rate and fractionation parameters were identical to Jnkl purification.
  • p38delta For purification of p38delta, cells were grown at 37 °C to OD600 1.0 and induced with 0.5 mM IPTG for 2 hours. MCAC fractions were dialysed against Buffer B plus 100 mM KC1, then diluted threefold with Buffer B immediately before loading on a 1 ml HiTrap SP column. The column was washed with 5 column volumes of Buffer B plus 50 mM KC1, and eluted with 10 column volumes to Buffer B plus 500 mM KC1, at a flow rate of 0.5 ml/min taking 0.5 ml fractions.
  • Mekl For purification of Mekl, a fresh transformation was grown for 12 hours at 30 °C, split 1:100 into fresh media, and grown to OD600 0.7 at 37 °C, then induced with 1 mM IPTG for 3 hours.
  • MCAC fractions were desalted on a PD-10 column into Buffer E (40 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT, 10% glycerol) and applied to a 1 ml HiTrap Q column. The column was washed with 10 CV Buffer E and Mekl was eluted in a single step of Buffer E plus 85 mM NaCl.
  • Buffer E 40 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM DTT, 10% glycerol
  • Mekl -DD Huand and Erikson, 1994
  • cells were grown to OD600 of 0.7 at 37C and induced with 0.5 mM IPTG for 3 hours. Purification conditions were identical to RhoA and H-ras.
  • Recombinant ERK and Mekl-DD were mixed in a 20:1 molar ratio in IX Kinase Activation Buffer (100 mM NaCl, 10 mM MgCl 2 , 10 mM HEPES, pH 8.0, 10% glycerol).
  • ATP was added to 1 mM and the mixture incubated for 30 minutes at 23 °C. Excess ATP was removed from the reaction by desalting on a PD-10 column into Buffer G (20 mM HEPES, pH 8.0, 2 mM DTT, 10% glycerol) plus 25 mM NaCl.
  • the eluate was loaded on a 1 ml HiTrap Q column and washed with 20 column volumes of Buffer G plus 100 mM ' NaCl.
  • ppERK was eluted with a 60 column volume gradient to Buffer G plus 300 mM NaCl at 0.5 ml/min, collecting 0.5 ml fractions.
  • Fractions containing ppERK were diluted six fold with buffer G and reapplied to a 1 ml HiTrapQ column.
  • the column was washed with 50 mM NaCl, and the ppERK was eluted with a single step of Buffer G plus 500 mM NaCl.
  • Example 11 In vitro selection of cis-hammerhead derived ERK and ppERK NASMs
  • Figure IB illustrates an RNA ribozyme library derived from a hammerhead sequence pool consisting of up to 10 17 variants of randomized sequences appended to the hammerhead ribozyme motif (Figure 19).
  • the ribozyme library is prepared on DNA synthesizer. Random nucleotides are incorporated during the synthesis to generate pools of roughly IO 17 molecules.
  • Figure 19 illustrates linker scanning library designed to identify cis-hammerhead NASMs that are modulated by the protein kinase ERK.
  • the linker library was generated by appending an ERK target modulation domain to the randomized linker domain to create a library of potential ERK-modulated cis-hammerhead NASMs.
  • the linker library of ERK-modulated cis-hammerhead NASMs consists of up to 65,000 variants. Most molecules in the randomized NASM pools are non-functional NASMs. Selection of NASMs. Sorting among the billions of NASMs to find the desired molecules starts from the complex sequence pool, whereby desired target-modulated NASMS are isolated through an iterative in vitro selection process: in addition to the target- activated ribozymes that one desires, the starting pool is usually dominated by either constitutively active or completely inactive ribozymes. The selection process removes both types of contaminants. In a following amplification stage, thousands of copies of the surviving sequences are generated to enable the next round of selection. During amplification, random mutations can be introduced into the copied molecules — this
  • linker library of ERK NASMs consists of up to 65,000 variants
  • the library consists of up to IO 17 variants of randomized sequences appended to the hammerhead ribozyme motif) of target modulated ribozymes containing randomized target modulation domains and randomized linker regions (1 ⁇ M)
  • RNA molecules (radiolabeled internally) were then removed from the mixture by electrophoresis through a 7M urea, 8% PAGE gel, followed by cutting the uncleaved band from the gel. Detection of cleaved and uncleaved RNAs were carried out using a phosphor- imager system. The uncleaved hammerhead-derived RNAs were then subjected to a round of positive selection for ERK, or ppERK, target modulation, by incubating the pool of RNA in selection to which the protein target (50nM to 1 ⁇ M ERK, or 50nM to 1 ⁇ M ppERK) were added, and the mixture was further incubated at 37 °C for 30 minutes.
  • the protein target 50nM to 1 ⁇ M ERK, or 50nM to 1 ⁇ M ppERK
  • RNA molecules (radiolabeled internally) were then isolated and removed from the mixture by electrophoresis through a 7M urea, 8% PAGE gel, followed by cutting the cleaved band from the gel. incubated with reverse transcription mix at 65 °C for 1 hour using a thermostable reverse transcriptase. The pool was subsequently separated from the reverse transcription mixture by filtration and was then amplified using PCR first with the substrate 3-specific 5'-PCR primer and the library-specific 3'-PCR primer, and secondly with the regeneration 5'-PCR primer and the library-specific 3'-PCR primer in order to add the T7 promoter.
  • Cleavage Assays Cleavage assays were performed using radiolabeled RNA and analytical denaturing polyacrylamide gel elecrophoresis (PAGE) (gel-based assays). Assays were performed upon both the library and clonal sequences. In a representative gel-based assay transcription was performed in the presence of alpha- 32 P-labelled UTP, and the resultant transcripts were gel-purified using denaturing PAGE.
  • Assay mixtures were then made in which 10 ⁇ l reactions containing RNA (lu ⁇ M), and protein target (1 ⁇ M) were incubated at 37 °C for between 15min and 16h in a reaction buffer containing buffer containing buffer containing KC1 (0- 150 mM), HEPES pH 7.5 (20 mM), MgCl 2 (20 mM), EDTA (0.5 mM).
  • the resultant samples are quenched by the addition of EDTA and the relative extents of cleavage measured by comparison of the intensity of the corresponding bands on a denaturing PAGE observed by reading a phosphorimager plate that had been exposed to the gel.
  • Example 12 Nucleic acid sensor molecules modulated by ERK and phosphorylated ERK generated by engineering target modulation domains into hammerhead catalytic domains.
  • ERK and ppERK modulated nucleic acid sensor molecules were generated by a strategy combining both engineering and in vitro selection, Figure 19.
  • Target modulation domains selected for binding to ERK and to the phosphorylated form of ERK (ppERK) are the starting point of the engineering efforts (Seiwert et al. 2000). These target modulation domains when isolated as discrete aptamers specifically recognize ERK but do not detectably interact with other mitogen-activated protein kinases such as Jun N-terminal kinase or p38 (as monitored by the ability to inhibit kinase activity).
  • An aptamer (target modulation domain) selected for ppERK binding efficiently discriminates between phosphorylated and non-phosphorylated forms of the protein, binding ppERK with a K ⁇ > of 4.7 nM and ERK with a K D of 50 nM (Seiwert et al. 2000).
  • NASMs e.g., certain ribozyme NASMs, derived through direct engineering, when assayed as described above, exhibit little or no cleavage activity in the absence of ERK and no detectable ERK- modulation of cleavage activity in the presence of ERK.
  • Inactive NASMs remained unresponsive to added ERK protein in concentrations ranges tested from 50 nM to 5 uM.
  • Ligated RNA molecules were then removed from the mixture by incubation with immobilized neutravidin. The flow through was collected by filtration and to this was added more substrate 3 (2 ⁇ M), protein target (1 ⁇ M ERK or ppERK) and RNase inhibitor were added and the mixture was further incubated at 25 °C for between 15 minutes and 1 hour. Ligated RNA molecules were then captured by incubation with immobilized neutravidin and, after washing, the matrix was incubated with reverse transcription mix at 65 °C for 1 hour using a thermostable reverse transcriptase.
  • the matrix was subsequently separated from the reverse transcription mixture by filtration and was then amplified using PCR first with the substrate 3-specific 5' -PCR primer and the library- specific 3' -PCR primer, and secondly with the regeneration 5' -PCR primer and the library- specific 3'-PCR primer in order to add the T7 promoter. Transcriptions were performed with these PCR products directly and after denaturing PAGE purification the entire process was repeated for several rounds with assays run after every round. Ligation Assays
  • Ligation assays were performed using radiolabeled RNA and analytical denaturing polyacrylamide gel elecrophoresis (PAGE) (gel-based assays), or by PCR utilizing the substrate 3-specific 5'-PCR primer (PCR-based assay). Assays were performed upon both the library and clonal sequences. In a representative gel-based assay transcription was performed in the presence of alpha- 32 P-labelled UTP, and the resultant transcripts were gel- purified using denaturing PAGE.
  • PAGE polyacrylamide gel elecrophoresis
  • Assay mixtures were then made in which 10 ⁇ l reactions containing RNA (lu ⁇ M), effector oligonucleotide (1.5 ⁇ M), substrate 3 (5 ⁇ M) and optionally RNase-inhibitor and protein target (1 ⁇ M) were incubated at 25 °C for between 15min and 16h in a reaction buffer containing protein target (1 ⁇ M ERK or ppERK), KC1 (150 mM), HEPES (20 mM), MgC12 (10 mM), EDTA (1 mM), DTT (1 mM), tRNA (0.1 mg/ml) and glycerol (10% w/v) .
  • the resultant samples were quenched by the addition of EDTA and the relative extents of ligation were measured by comparison of the intensity of the corresponding bands on a denaturing PAGE observed by reading a phosphorimager plate that had been exposed to the gel.
  • PCR assays were performed using the same reaction mixture except that the relative extents of ligation in different samples were compared by observing the relative rates of appearance of PCR products using the substrate 3-specific 5'- PCR primer and the library-specific 3'-PCR primers.
  • the detailed methods for the analysis ERK-modulated NASM clones is given in Example IA, Cloning, sequencing and characterization of individual NASMs.
  • ERK-dependent ligase ribozymes were built on the catalytic core of the LI ligase ribozyme of Robertson and Ellington (2000; NAR 28, 1751-1759) in which the non- conserved, stem C element was replaced with the ERK-interacting domain and joined to the catalytic core by a 2-4 base pair helical element, or "communication module”.
  • Ten different ribozymes were designed, identical in sequence except for the bases in the linker domain ( Figure 32). (SEQ ID NOs: 109- 116) Templates for transcription by T7 RNA polymerase were prepared for each ribozyme by PCR amplification using a set of three overlapping primer oligonucleotides. The forward, or 5' primer
  • TAATACGACTCACTATAGGACTTCGGCGAAAGCCGTTCGACC included the T7 RNA polymerase promoter and sequence corresponding to the 5 '-proximal region of the LI ligase catalytic core.
  • the other two primers corresponded to sequences spanning the 3'-proximal portion of the LI ligase core
  • RNA was synthesized by in vitro transcription (Milligan & Uhlenbeck (1989) Methods Enzymol 180, 51-62) using the T7-MEGAshortscriptTM transcription kit from Ambion, and purified by gel-filtration (to remove transcription buffer components) and/or polyacrylamide gel electrophoresis (PAGE). The purified RNAs were then quantified by their absorbance at 260 nm, and stored in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at -20 °C. Radiolabeled RNAs were prepared exactly as described above, except that ⁇ - P-UTP was included in the in vitro transcription reaction.
  • ppERK-dependent ligase ribozymes were built on the catalytic core of the LI ligase ribozyme of Robertson and Ellington (2000; NAR 28, 1751-1759) in which the non- conserved, stem C element was replaced with the ERK-interacting domain and joined to the catalytic core by a 2-4 base pair helical element, or "communication module”.
  • Fourteen different ribozymes were designed, identical in sequence except for the bases in the linker domain ( Figure 41) (SEQ ID NO:352). Templates for transcription by T7 RNA polymerase were prepared for each ribozyme by PCR amplification using a set of three overlapping primer oligonucleotides. The forward, or 5' primer
  • TAATACGACTCACTATAGGACTTCGGCGAAAGCCGTTCGACC included the T7 RNA polymerase promoter and sequence corresponding to the 5 '-proximal region of the LI ligase catalytic core.
  • the other two primers corresponded to sequences spanning the 3 '-proximal portion of the LI ligase core
  • RNA was synthesized by in vitro transcription (Milligan & Uhlenbeck (1989) Methods Enzymol 180, 51-62) using the T7-MEGAshortscriptTM transcription kit from Ambion, and purified by gel-filtration (to remove transcription buffer components) and/or polyacrylamide gel electrophoresis (PAGE). The purified RNAs were then quantified by their absorbance at 260 nm, and stored in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at -20 °C. Radiolabeled RNAs were prepared exactly as described above, except that ⁇ - 32 P-UTP was included in the in vitro transcription reaction.
  • Templates for transcription were generated by PCR, and the transcripts thus generated were also purified by denaturing (8M urea) polyacrylamide gel electrophoresis (PAGE). Gel-purification is followed by the localization of nucleic acids within the gel by UV-shadowing, nucleic-acid containing gel pieces are excised and the purified nucleic acids are recovered by electroelution.
  • the following synthetic oligonucleotides were utilized:
  • Substrate 3 (DNA, RNA underlined) Biotin-TEG-CATGCGACCTTACGATCAGATGACCTUGCACU (SEQ ID NO: 1
  • Substrate 3-specific 5' -PCR primer CATGCGACCTTACGATCAGAT (SEQ ID NO:322)
  • ERK selection-specific oligonucleotides Library (DNA) (random regions 3-5 nucleotides in length)
  • Example 17 Nucleic acid sensor molecules modulated by native ERK enzyme.
  • Hammerhead-derived nucleic acid sensor molecules (88 nucleotides in length) were selected from populations of nucleic acid molecules with randomized linker domain in stem II, as shown in Figure 19.
  • the Table in Figure 19 depicts illustrative linker-domain sequences of several of the ERK-modulated nucleic acid sensor molecules isolated from in vitro selection linker randomized clones (described in detail in Example 11). Each NASM displays a varying degree of modulation driven by addition of equal amounts (1 uM) native ERK. Individual ERK-modulated cis-hammerhead nucleic acid sensor molecules are shown in Figure 19.
  • Clones 1-14, 1-13, 1-2, 1-6, 2-7, 2-2, 2-3, 2-13, 2-14, and 2-20 were tested in target modulation assays as described previously.
  • the NASM's relative dependence on ERK is denoted in the Table in Figure 19 by the extent of activity of the ribozyme in the presence of ERK protein.
  • the time course of signal generation in the presence of nonphosphorylated ERK, phosphorylated ERK, and in the absence of protein is determined by measuring signal released over time by a radiolabeled nucleic acid sensor molecule. Significant amounts signal, corresponding to cleavage of the nucleic acid sensor molecule is observed over time only with the nonphosphorylated ERK.
  • Clones 1-2, 1-13 and 1-14 all display sensitivity to ERK concentration, as shown in Figures 20 A, B and C. However, of these three clones, clone 1-14 displays the greatest enhancement in activity upon addition of ERK. Clone 1-14 is able to differentiate between varying concentrations of ERK, as indicated by the dose- dependent change in the activity of clone-14 upon the addition of 5, 10, 20, 50 and 100 nM ERK ( Figure 21).
  • Example 18 ERK-modulated NASM-based competitive inhibition assays, & target- protein specific profiling biosensors.
  • the specificity of the interaction between clone 1-14 and ERK was assessed by measuring the activity of clone 1-14 in the presence of 50 nM ERK and increasing concentrations of known protein kinase inhibitors.
  • the kinase inhibitors staurosporine and 5-iodo-tubercidin are compounds that are known bind to in an ATP-substrate competitive manner to ERK and to modulate ERK kinase catalytic activity, decreasing its ability to phosphorylate other proteins.
  • kinase inhibitors would compete directly for the ATP-binding site in ERK, and thereby block NASM activation by ERK.
  • FIG. 58 shows additional staurosporine competitive NASM assay results using an LI -ligase-derived NASM which is also modulated by ERK protein. Additional modifications of the 3-piece Ll-ligase NASM (cf, Figure 36, constructs 27 and constructs 28) render these biosensors suitable for cellular assays as intracellular biosensors (c , Figure 37 and the detailed description of the 3-piece, and 1-piece ligases described in detail in Example 22). Thus, ERK-modulated nucleic acid sensor molecules are used for cell- based drug discovery and drug candidate screening.
  • ERK NASMs (clone 1-14 and constructs 27 and 28) are modulated specifically by ERK only in its fully active, and not by ppERK, or by related homologues and MAP Kinase pathway molecules, For example clone 1-14 recognizes only ERK, and not related kinase MEK.
  • NASMs and more specifically, the ERK and ppERK NASMs are shown here to be useful specific protein profiling biosensors.

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Abstract

La présente invention concerne des méthodes de production par génie génétique d'une molécule de sonde d'acide nucléique. Des biocapteurs comprennent une pluralité de molécules de sonde d'acide nucléique marquées avec des premier et deuxième fragments de signalisation. Les molécules de sonde d'acide nucléique reconnaissent les molécules cibles qui ne se lient pas naturellement à l'ADN. La liaison d'une molécule cible aux molécules de sonde provoque un changement au voisinage des fragments de signalisation qui entraîne un changement des propriétés optiques des molécules de sonde d'acide nucléique situées sur le biocapteur. Cette invention concerne également des réactifs et des systèmes permettant de mettre en oeuvre le procédé. Le présent procédé est utile dans des applications de diagnostic et d'optimisation des médicaments.
PCT/US2002/025319 2001-08-09 2002-08-09 Molecules de sonde d'acide nucleique et methodes d'utilisation de ces dernieres Ceased WO2003014375A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1552002A4 (fr) * 2002-06-18 2006-02-08 Archemix Corp Molecules toxine-aptamere et procedes d'utilisation correspondants
US7960102B2 (en) 2002-07-25 2011-06-14 Archemix Corp. Regulated aptamer therapeutics
US8039443B2 (en) 2002-11-21 2011-10-18 Archemix Corporation Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics
WO2014102806A1 (fr) * 2012-12-31 2014-07-03 Yeda Research And Development Co. Ltd. Biocapteurs protéiniques, réseaux de capteurs à réaction croisée et procédés d'utilisation correspondants
US10100316B2 (en) 2002-11-21 2018-10-16 Archemix Llc Aptamers comprising CPG motifs
US10557851B2 (en) 2012-03-27 2020-02-11 Ventana Medical Systems, Inc. Signaling conjugates and methods of use

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* Cited by examiner, † Cited by third party
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US8853376B2 (en) 2002-11-21 2014-10-07 Archemix Llc Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics

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ES2176233T3 (es) * 1992-12-04 2002-12-01 Univ Yale Deteccion diagnostica amplificada con ribozimas.
MX9706501A (es) * 1995-02-27 1998-02-28 Intelligene Ltd Deteccion de biomoleculas.
JP4318856B2 (ja) * 1998-03-05 2009-08-26 ジョンソン・アンド・ジョンソン・リサーチ・ピー・ティー・ワイ・リミテッド チモーゲン性核酸検出方法、および関連分子およびキット
DE19811618C1 (de) * 1998-03-17 2000-08-31 Andreas Jenne Ribozym codierende DNA und ein Oligonucleotidsubstrat enthaltende Zusammensetzung und Verfahren zur Messung von Transkriptionsraten
EP1066312A4 (fr) * 1998-03-28 2002-01-02 Univ Utah Res Found Ribozymes a tete de marteau et leurs derives dits circulaires, en epingle a cheveux, circulaires/en epingle a cheveux, en lasso, en epingle a cheveux/lasso

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1552002A4 (fr) * 2002-06-18 2006-02-08 Archemix Corp Molecules toxine-aptamere et procedes d'utilisation correspondants
US7960102B2 (en) 2002-07-25 2011-06-14 Archemix Corp. Regulated aptamer therapeutics
US8039443B2 (en) 2002-11-21 2011-10-18 Archemix Corporation Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics
US10100316B2 (en) 2002-11-21 2018-10-16 Archemix Llc Aptamers comprising CPG motifs
US10557851B2 (en) 2012-03-27 2020-02-11 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
US11906523B2 (en) 2012-03-27 2024-02-20 Ventana Medical Systems, Inc. Signaling conjugates and methods of use
WO2014102806A1 (fr) * 2012-12-31 2014-07-03 Yeda Research And Development Co. Ltd. Biocapteurs protéiniques, réseaux de capteurs à réaction croisée et procédés d'utilisation correspondants
US9696310B2 (en) 2012-12-31 2017-07-04 Yeda Research And Development Co. Ltd. Molecular sensor and methods of use thereof
US10281459B2 (en) 2012-12-31 2019-05-07 Yeda Research And Development Co. Ltd. Protein biosensors, cross reactive sensor arrays and methods of use thereof

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