HK1125678B - Non-fluorescent energy transfer - Google Patents

Description

Non-fluorescent energy transfer

Technical Field

The present invention relates to the field of molecular biology and biomolecular chemistry. In certain embodiments, reagents and assays involving non-fluorescent energy transfer are provided.

Background

Techniques for obtaining information about biomolecules, such as nucleic acids and proteins, find application in many different disciplines, including various branches of medical science. Many of these techniques involve the use of fluorescent labels (label) to generate a detectable signal. For example, one class of fluorescent dyes that has been developed includes energy transfer fluorescent dyes. In general, the energy transfer processes involving these dyes involve dipole-dipole resonance interactions between a donor moiety (donor moiety) and an acceptor moiety (acceptor moiety) in the same or different biomolecules. In these processes, energy emitted from the donor moiety is absorbed by the acceptor moiety when the donor moiety and the acceptor moiety are positioned in close enough proximity and properly oriented with respect to each other. When this absorbed energy causes the acceptor moiety to fluoresce, a detectable signal is generated.

Exemplary nucleic acid analysis methods that typically employ energy transfer fluorescent dyes include hybridization-based assays such as nucleic acid amplification procedures (e.g., Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), nucleic acid sequence-based extension (NASBA), and Ligase Chain Reaction (LCR)), high-density nucleic acid array-based methods, Single Nucleotide Polymorphism (SNP) analysis, and nucleic acid sequencing techniques. Further, various methods are known for the determination of other types of biomolecules that can use energy transfer to effect detection. For example, proteins can be detected and quantified using various techniques, including SDS-polyacrylamide gel electrophoresis, capillary electrophoresis, enzymatic assays, cell-based assays, and various immunological techniques such as Western blotting and ELISA.

In addition, several diagnostic and analytical assays have been developed which involve the detection of multiple components in a sample with fluorescent dyes, including, for example, flow cytometry (Lanier et al, (1984) 'human lymphocyte sub-screening by using the same gene-chromatography of luminescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression (the subpopulations of human lymphocytes identified by immunofluorescence and flow cytometry analysis: Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression)',J.Immunol.132: 151-: one and two parameter flow cytometry (high resolution chromosome analysis: one or two parameter flow cytometry) ",Chromosoma73: 9-27) and many of the above assays. For these assays, a set of two or more spectrally resolvable (fluorescent) dyes needs to be used simultaneously in order to be able to detect more than one target substance in a sample simultaneously. The use of multiple dyes to simultaneously detect multiple components in a sample reduces the time required to sequentially detect each component in the sample. In the case of multi-locus DNA probe assays, the use of multiple spectrally resolvable fluorescent dyes can reduce the number of reaction tubes requiredThe number of the reagents, thereby simplifying the experimental scheme and facilitating the manufacture of the kit for specific applications. For example, in the case of automated DNA sequencing, the use of multiple spectrally resolvable fluorescent dyes allows analysis of all four bases in a single lane, thereby increasing throughput (throughput) over the single color method, eliminating uncertainty due to differences in electrophoretic mobility between lanes.

Among various multiplex detection methods (multiplex detection methods), multiplex PCR detection using 5' nuclease probes, molecular beacons, FRET probes, or hybridization probes typically involves pooling (posing) of quenched or unquenched fluorescent probes, e.g., to improve assay throughput relative to protocols that employ a single probe in a given reaction. For example, multiplex assays are commonly used as part of diagnostic procedures to detect multiple genotype markers (markers) or pathogens in samples obtained from patients. In these assay formats, as the number of probes in the reaction mixture increases, the overall baseline or background fluorescence from the pooled probes also increases cumulatively. This baseline fluorescence also increases in almost all assay systems as the amount of any single probe increases. Baseline fluorescence typically adversely affects the performance of a given assay, for example, by reducing the detection sensitivity and dynamic range of the assay. Thus, baseline fluorescence actually limits the total number of fluorescent probes and/or the amount of a given probe that can be used at one time in a particular assay.

Summary of The Invention

The present invention provides biomolecules and other reagents that involve non-fluorescent energy transfer. For example, certain biomolecules comprising substantially non-fluorescent donor moieties described herein can be used to effect detection of a target biomolecule. There are several advantages to using these donor moieties for such detection, one of which is a reduction in background fluorescence relative to methods employing fluorescent donor moieties. In addition to providing reaction mixtures and methods, the present invention also provides related kits and systems.

In one aspect, the invention provides biomolecules comprising at least one substantially non-fluorescent donor moiety capable of transferring non-fluorescent energy to at least one acceptor moiety or reporter moiety (e.g., a fluorescent dye) when the acceptor moiety or reporter moiety is sufficiently close thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy. The biomolecule typically comprises at least one nucleoside, at least one amino acid, at least one sugar and/or at least one lipid. In certain embodiments, the biomolecule comprises an acceptor moiety and/or at least one quencher moiety (quencher). In some embodiments, the substantially non-fluorescent donor moiety and/or acceptor moiety is attached to the biomolecule through at least one linker moiety.

The biomolecules described herein include many different embodiments. For example, in certain embodiments, the biomolecule comprises a biopolymer synthesis reagent (e.g., phosphoramidite). In some embodiments, the biomolecule comprises a biopolymer. In these embodiments, the different monomeric units of the biopolymer optionally comprise substantially non-fluorescent donor moieties and acceptor moieties. In certain of these embodiments, the different monomeric units of the biopolymer comprise both a substantially non-fluorescent donor moiety and an acceptor moiety. Optionally, the substantially non-fluorescent donor moiety and the acceptor moiety are not connected to each other by at least one linker moiety. By way of further example, in certain embodiments, the biomolecule comprises at least one oligonucleotide or at least one polynucleotide. Oligonucleotides typically comprise primer nucleic acids or probe nucleic acids (e.g., hybridization probes, 5' -nuclease probes, and hairpin probes). In some embodiments, the biomolecule comprises at least one peptide, at least one polypeptide, at least one protein, at least one enzyme, at least one hormone, or at least one immunoglobulin.

In another aspect, the invention provides a reaction mixture comprising at least one nucleotide (e.g., an extendable nucleotide and/or a terminator nucleotide), at least one primer nucleotide, and/or at least a first probe nucleic acid (e.g., a hybridization probe, a 5' -nuclease probe, and a hairpin probe), wherein one or more of the nucleotide, primer nucleic acid, or first probe nucleic acid comprises at least one substantially non-fluorescent donor moiety that is capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy. In some embodiments, the nucleotide, primer nucleic acid, or first probe nucleic acid comprises an acceptor moiety (e.g., a fluorescent dye). Optionally, the nucleotide, primer nucleic acid, or first probe nucleic acid comprises at least one quencher moiety. In certain embodiments, the substantially non-fluorescent donor moiety and/or acceptor moiety is linked to the nucleotide, primer nucleic acid, or first probe nucleic acid through at least one linker moiety. In some embodiments, the reaction mixture further comprises at least one biocatalyst that catalyzes the incorporation of nucleotides. Optionally, the substantially non-fluorescent donor moiety is linked to the nucleotide, the primer nucleic acid, or the first probe nucleic acid through at least one linker moiety. In certain embodiments, the reaction mixture includes at least a second probe nucleic acid (e.g., optionally used as a hybridization probe in some embodiments) that comprises an acceptor moiety. In some of these embodiments, the second probe nucleic acid further comprises at least one quencher moiety. For example, this reaction mixture may be used to perform various real-time PCR monitoring schemes or nucleic acid sequencing procedures, as well as for many other possible applications.

In another aspect, the invention provides a reaction mixture comprising at least a first biopolymer synthesis reagent comprising a biomolecule of at least one substantially non-fluorescent donor moiety capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy. In some embodiments, the substantially non-fluorescent donor moiety is attached to the first biopolymer synthesis reagent via at least one linker moiety. In certain embodiments, the first biopolymer synthesis reagent comprises an acceptor moiety, the substantially non-fluorescent donor moiety and the acceptor moiety not being connected to each other by at least one linker moiety. In other embodiments, the first biopolymer synthesis reagent comprises an acceptor moiety, the substantially non-fluorescent donor moiety and the acceptor moiety being connected to each other by at least one linker moiety. Optionally, the solid support (solid support) comprises a first biopolymer synthesis reagent. In some embodiments, the reaction mixture comprises at least a second biopolymer synthesis reagent comprising an acceptor moiety and/or at least one quencher moiety. By way of further example, in certain embodiments, the first biopolymer synthesis reagent comprises a polypeptide synthesis reagent, while in other embodiments, the first biopolymer synthesis reagent comprises a nucleic acid synthesis reagent (e.g., a phosphoramidite). This reaction mixture is optionally used for example for the synthesis and/or labeling of biopolymers.

In another aspect, the invention provides a method of detecting a target biomolecule. The method comprises (a) binding at least one probe biomolecule (e.g., an immunoglobulin) to a target biomolecule, wherein the probe biomolecule comprises at least one substantially non-fluorescent donor moiety and at least one acceptor moiety (e.g., a fluorescent dye) that accepts non-fluorescent energy transferred from the substantially non-fluorescent donor moiety and emits light in response to the accepted non-fluorescent energy. In addition, the method includes (b) detecting light emitted from the receptor moiety, thereby detecting the target biomolecule. In some embodiments, the target biomolecule comprises a target nucleic acid, and thus the method comprises amplifying at least a subsequence of the target nucleic acid prior to and/or during (b). Typically, the probe biomolecule comprises a biopolymer. In certain embodiments, the different monomeric units of the biopolymer comprise a substantially non-fluorescent donor moiety and an acceptor moiety. The different monomeric units of the biopolymer optionally comprise both a substantially non-fluorescent donor moiety and an acceptor moiety, which are typically connected to each other by at least one linker moiety or not. By way of further example, in certain embodiments, the probe (e.g., hybridization probe, 5' -nuclease probe, and hairpin probe) and/or the target biomolecule comprises a nucleic acid. In these embodiments, (a) generally comprises hybridizing the probe and the target biomolecule.

In another aspect, the invention provides another method of detecting a target biomolecule. The method comprises (a) providing at least a first and a second probe biomolecule, wherein the first probe biomolecule comprises at least one substantially non-fluorescent donor moiety, and wherein the second probe biomolecule comprises at least one acceptor moiety (e.g. a fluorescent dye). The method further includes (b) binding the first and second probe biomolecules to the target biomolecule such that the acceptor moiety accepts non-fluorescent energy transferred from the substantially non-fluorescent donor moiety and emits light in response to the accepted non-fluorescent energy. In addition, the method includes (c) detecting light emitted from the receptor moiety, thereby detecting the target biomolecule. In some embodiments, the substantially non-fluorescent donor moiety on the first probe biomolecule is an isothiocyanate other than 4-dimethylaminophenylazophenyl-4' -isothiocyanate (DABITC). In some embodiments, the target biomolecule comprises a target nucleic acid, the method comprising amplifying at least a subsequence of the target nucleic acid prior to and/or during (c). In certain embodiments, the first and/or second probe biomolecule comprises an immunoglobulin. In some embodiments, the first probe biomolecule, the second probe biomolecule, and/or the target biomolecule comprise a nucleic acid. In these embodiments, the first and/or second probe biomolecule typically comprises a hybridization probe, a 5' -nuclease probe, and a hairpin probe. In some of these embodiments, (b) comprises hybridizing the first probe biomolecule, the second probe biomolecule, and the target biomolecule.

In another aspect, the invention provides methods of performing proximity assays (e.g., lipid mixing assays). The method comprises (a) providing at least a first and a second probe biomolecule, wherein the first probe biomolecule comprises at least one substantially non-fluorescent donor moiety, and wherein the second probe biomolecule comprises at least one acceptor moiety. The method further includes (b) placing the first and second probe biomolecules in a relative to one antenna first position at which the first and second probe biomolecules are sufficiently close to each other that the acceptor moiety accepts non-fluorescent energy transferred from the substantially non-fluorescent donor moiety and emits light in response to the accepted non-fluorescent energy. In addition, the method further comprises (c) moving the first and second probe biomolecules to at least a second, relatively speaking location, and (d) monitoring light emitted from the receiving body before, after and/or while the first and second probe biomolecules are moved to the second location, thereby performing the proximity determination. Typically, the first and/or second probe biomolecules comprise an oligonucleotide, an immunoglobulin or a lipid.

In another aspect, the invention provides a method of extending a primer nucleic acid. The method comprises incubating a target nucleic acid with (a) at least one extendable nucleotide and/or at least one terminator nucleotide, (b) at least one biocatalyst that catalyzes nucleotide incorporation and (c) at least one primer nucleic acid that is at least partially complementary to at least a subsequence of the target nucleic acid under conditions such that the biocatalyst that catalyzes nucleotide incorporation can extend the primer nucleic acid by incorporating the extendable nucleotide and/or the terminator nucleotide at an end of the extended primer nucleic acid to produce at least one extended primer nucleic acid, wherein the primer nucleic acid, the extendable nucleotide and/or the terminator nucleotide comprises at least one substantially non-fluorescent donor moiety that is capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is sufficiently proximal thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy, thereby extending the primer nucleic acid. In certain embodiments, a plurality of extended primer nucleic acids are produced, the method comprising identifying a terminator nucleotide in the extended primer nucleic acid, whereby at least a portion of the base sequence of the target nucleic acid is determinable from the identified terminator nucleotide. In some embodiments, the primer nucleic acid, the extendable nucleotide, and/or the terminator nucleotide comprises an acceptor moiety. In certain embodiments, the substantially non-fluorescent donor moiety and the acceptor moiety are not connected to each other by at least one linker moiety, while in other embodiments, the substantially non-fluorescent donor moiety and the acceptor moiety are connected to each other by at least one linker moiety.

In another aspect, the invention provides a method of producing a phosphoramidite. The method comprises (a) attaching at least one substantially non-fluorescent donor moiety to a compound comprising at least one protecting group. The substantially non-fluorescent donor moiety is capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is sufficiently close thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy. The method further comprises (b) attaching a group comprising the formula:

wherein R is₁And R₂Is an alkyl group independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and heptyl; r₃Is (CH)₂)₂CN or CH₃Thereby producing a phosphoramidite. Exemplary protecting groups are selected from, for example, trityl (trityl), monomethoxytrityl, dimethoxytrityl, Jerusalem artichoke glycosyl (levulinyl), fluorenylmethoxycarbonyl and benzhydryloxycarbonyl. In some embodiments, the phosphoramidite comprises the following formula:

in another aspect, the invention provides a business method comprising (a) receiving a purchase order from a customer for at least a first biomolecule comprising at least one substantially non-fluorescent donor moiety capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto such that the acceptor moiety emits light in response to the received non-fluorescent energy. In addition, the business method includes (b) supplying the first biomolecule to the customer in accordance with the purchase order.

In another aspect, the invention provides a kit comprising at least a first biomolecule comprising at least one substantially non-fluorescent donor moiety capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy. In some embodiments, the solid support comprises a first biomolecule. In certain embodiments, the first biomolecule comprises an acceptor moiety. In some of these embodiments, the substantially non-fluorescent donor moiety and the acceptor moiety are not connected to each other by at least one linker moiety, while in other embodiments, the substantially non-fluorescent donor moiety and the acceptor moiety are connected to each other by at least one linker moiety. In certain embodiments, the kit comprises a second biomolecule comprising an acceptor moiety. Optionally, the kit includes, for example, one or more of buffers, salts, metal ions, a biocatalyst that catalyzes nucleotide incorporation, pyrophosphatase, glycerol, dimethyl sulfoxide, and/or poly rA. Typically, the kit comprises at least one container for packaging at least a first biomolecule.

The kit includes multiple embodiments that can be used to perform a number of different applications. For example, in some embodiments, the first biomolecule comprises an immunoglobulin and the kit comprises instructions for binding the immunoglobulin to a target epitope and detecting binding of the immunoglobulin to the target epitope. Optionally, the first biomolecule comprises a lipid and the kit includes instructions for performing a proximity assay with the lipid. In certain embodiments, the first biomolecule comprises a primer nucleic acid and the kit includes instructions for extending the primer nucleic acid. In some embodiments, the first biomolecule comprises an extendable nucleotide and/or a terminator nucleotide. In certain embodiments, the first biomolecule comprises a first biopolymer synthesis reagent and the kit includes instructions for synthesizing a biopolymer using the first biopolymer synthesis reagent. In some of these embodiments, the first biopolymer synthesis reagent comprises a polypeptide synthesis reagent. Optionally, the kit comprises at least a second biopolymer synthesis reagent. In some embodiments, the second biopolymer synthesis reagent comprises an acceptor moiety. In certain embodiments, the first biopolymer synthesis reagent comprises a nucleic acid synthesis reagent (e.g., a phosphoramidite). In some embodiments, the first biomolecule comprises at least a first probe nucleic acid. For example, the first probe nucleic acid optionally comprises a hybridization probe, a 5' -nuclease probe, or a hairpin probe. In some of these embodiments, the kit comprises a nucleic acid comprising at least a second probe comprising an acceptor moiety. In certain embodiments, this second probe nucleic acid is used as a hybridization probe in conjunction with the first probe nucleic acid to perform various hybridization probe assays.

In another aspect, the invention provides a system comprising (a) at least one container and/or solid support comprising at least one biomolecule comprising at least one substantially non-fluorescent donor moiety capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy. The system also includes (b) at least one radiation source positioned to direct electromagnetic radiation at the donor portion. Additionally, the system includes (c) at least one detection device positioned to detect light emitted from the receptor portion when the receptor portion is sufficiently proximate to the substantially non-fluorescent donor portion. Typically, the system includes at least one logic device operatively connected to the detection device, the logic device containing one or more instruction sets for scaling (scale) the detected light emission from the recipient portion. In some embodiments, the system comprises (d) at least one thermal regulator capable of forming thermal communication with the container and/or the solid support to regulate the temperature proximate to the container and/or the solid support, and/or (e) at least one fluid transfer device capable of transferring fluid to and/or from the container and/or the solid support.

The substantially non-fluorescent donor moieties in the various biomolecules, reaction mixtures, methods, kits, and systems described herein include various embodiments. For example, in some embodiments, the substantially (substitially) non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorescein, or 5-carboxy-amino pentachlorofluorescein. In certain embodiments, the peak visible absorptions of the substantially non-fluorescent donor moiety and the acceptor moiety differ by about 100nm or greater. Typically, the ratio of detectable absolute fluorescent emission from the 6-carboxyfluorescein moiety to detectable absolute fluorescent emission from the latter is about 1000: 1 or greater at substantially the same concentration as the substantially non-fluorescent donor moiety. In certain embodiments, the substantially non-fluorescent donor moiety is carboxyfluorescein, 1, 2-stilbene-2, 2' -disulfonic acid, or an isothiocyanate. In other embodiments, the substantially non-fluorescent donor moiety is an isothiocyanate other than 4-dimethylaminophenylazophenyl-4' isothiocyanate (DABITC).

In some embodiments, a substantially non-fluorescent donor moiety and an acceptor moiety described herein are not connected to each other by a linker moiety. In other embodiments, the substantially non-fluorescent donor moiety and the acceptor moiety described herein are connected to each other by a linker moiety. In some of these embodiments, the linker moiety lacks the following structure:

wherein R is₄To a C which is attached to a substantially non-fluorescent donor moiety_1-5An alkyl group; r₅Selected from NH, S and O; r₆Selected from the group consisting of alkenes, dienes, alkynes, and five or six membered rings having at least one unsaturated bond or a fused ring structure attached to a carbonyl carbon; r₇Comprising a functional group that connects the linker moiety to the acceptor moiety.

Brief Description of Drawings

FIGS. 1A-F illustrate certain representative embodiments of some agents of the invention.

FIG. 2 shows certain steps performed in a 5' -nuclease reaction according to one embodiment of the invention.

FIG. 3 shows some steps performed in an assay involving hairpin probes according to one embodiment of the invention.

FIG. 4 illustrates certain steps performed in an assay involving hybridization probes according to one embodiment of the present invention.

FIG. 5 illustrates certain steps performed in an assay involving labeled primers according to one embodiment of the present invention.

Figure 6 illustrates some steps performed in an assay involving a labeled protein according to certain embodiments of the invention.

Figure 7 shows some steps performed in a lipid mixing assay according to one embodiment of the present invention.

Figure 8 is a block diagram illustrating certain steps performed in a business method in accordance with one embodiment of the present invention.

FIG. 9 is a block diagram showing a representative system for performing proximity determination.

FIG. 10 is a graph of overlapping spectra obtained from oligonucleotides labeled with 6-carboxyfluorescein (or 6-FAM), 4 ', 5' -dimethoxy-5-carboxyfluorescein (or 5-Dmf), or 4 ', 5' -dimethoxy-6-carboxyfluorescein (or 6-Dmf).

FIG. 11 is an excerpt of a detail from the graph shown in FIG. 10, showing overlapping spectra obtained from excitation and emission scans of oligonucleotides labeled with 5-Dmf or 6-Dmf.

Figure 12 illustrates certain steps in a 5-and 6-DmF-DMT-CX-linker-phosphoramidite synthesis scheme according to one embodiment of the present invention.

FIG. 13 is a High Performance Liquid Chromatography (HPLC) trace showing detection of 5-Dmf and 6-Dmf.

FIG. 14 is an HPLC trace showing detection of pivaloyl 5-Dmf and pivaloyl 6-Dmf.

FIG. 15 is an HPLC trace showing detection of pivaloylated (pivalated) DmF + CX-linker.

FIG. 16 is an HPLC trace showing detection of Dmf-CX-linker.

Figure 17 is an HPLC trace showing detection of DmF-CX-linker-phosphoramidite.

Figure 18 is an HPLC trace showing detection of DmF-CX-linker-phosphoramidite.

FIG. 19 shows detection of Dmf-CX-linker-phosphoramidite³¹P NMR spectrum.

Figure 20 illustrates certain steps in a purification scheme to separate the 5-DmF isomer and the 6-DmF isomer from each other, according to one embodiment of the present invention.

FIG. 21 is a chromatogram showing detection of 5-Dmf.

FIG. 22 is a chromatogram showing detection of 6-Dmf.

Figure 23 illustrates certain steps in a 6-DmF-DMT-CX-linker-phosphoramidite synthesis scheme according to one embodiment of the present invention.

FIG. 24 is an HPLC trace showing detection of pivaloylated 6-Dmf.

FIG. 25 is an HPLC trace showing detection of 6-Dmf-CX-linker.

FIG. 26 is an HPLC trace showing detection of 6-Dmf-CX-linker-phosphoramidite.

Figure 27 illustrates certain steps in a 5-DmF-DMT-CX-linker-phosphoramidite synthesis scheme according to one embodiment of the present invention.

FIG. 28 is an HPLC trace showing detection of pivaloylation 5-Dmf.

FIG. 29 is an HPLC trace showing detection of 5-Dmf-CX-linker.

FIG. 30 is a graph of overlapping spectra obtained from excitation scans assayed for multiple hybridization probes including donor probes labeled with 6-FAM, 5-Dmf, or 6-Dmf.

FIG. 31 is a graph of the overlaid spectra of FIG. 30 normalized to the detected fluorescence excitation scan.

FIG. 32 is a graph of overlapping spectra obtained from emission scans assayed for multiple hybridization probes that include donor probes labeled with 6-FAM, 5-Dmf, or 6-Dmf, or lack donor probes.

FIG. 33 is a graph of overlapping spectra obtained from melting curve analysis of donor probes including 6-FAM on the label.

FIG. 34 is a graph of overlapping spectra obtained from melting curve analysis of donor probes including 5-Dmf on the label.

FIG. 35 is a graph of overlapping spectra obtained from kinetic PCR analysis (kinetic PCR analysis) including a 6-FAM-labeled donor probe.

FIG. 36 is a graph of overlapping spectra obtained from dynamic PCR analysis including donor probes labeled with 5-Dmf.

FIG. 37A shows a 5' -nuclease probe according to one embodiment of the invention.

Figure 37B shows a 5' -nuclease probe lacking a substantially non-fluorescent donor moiety.

FIG. 38 is a graph of overlapping spectra obtained from a PCR analysis in which a 5' -nuclease probe was used to monitor the reaction.

FIG. 39 is a graph of the overlaid spectra shown in FIG. 38 normalized for detected fluorescence.

FIG. 40 shows various donor and acceptor moieties in a representative hybridization probe pair.

FIG. 41A is a graph of spectra obtained from emission scans of hybridization probes labeled with LC-Red 610.

FIG. 41B is a graph of the spectrum obtained from the emission scan of the hybridization probe labeled JA-270.

FIG. 41C is a graph of the spectrum obtained from an emission scan of a hybridization probe labeled CY5.

FIG. 41D is a graph of the spectrum obtained from an emission scan of a hybridization probe labeled CY 5.5.

FIG. 42A is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probes labeled with FAM or DmF.

FIG. 42B is a graph of some of the overlapping spectra obtained from the emission scan shown at 42A, in which the fluorescence emission has been scaled.

FIG. 43A is a graph of overlapping spectra obtained from excitation scans of hybridization probe assays involving an acceptor probe labeled with LC-Red 610 and a donor probe labeled with FAM or Dmf.

FIG. 43B is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probe assays involving an acceptor probe labeled with LC-Red 610 and a donor probe labeled with FAM or DmF.

FIG. 43C is a graph of an overlapping spectrum obtained from the emission scan shown at 43B, in which the fluorescence emission has been scaled.

FIG. 44A is a graph of overlapping spectra obtained from excitation scans of hybridization probe assays involving acceptor probes labeled with JA-270 and donor probes labeled with FAM or DmF.

FIG. 44B is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probe assays involving acceptor probes labeled with JA-270 and donor probes labeled with FAM or DmF.

FIG. 44C is a graph of the overlaid spectra obtained from the emission scan shown at 44B, in which the fluorescence emission has been scaled.

FIG. 45A is a graph of overlapping spectra obtained from excitation scans of hybridization probe assays involving acceptor probes labeled with CY5 and donor probes labeled with FAM or Dmf.

FIG. 45B is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probe assays involving acceptor probes labeled with CY5 and donor probes labeled with FAM or DmF.

FIG. 45C is a graph of the overlaid spectra obtained from the emission scan shown at 45B, in which the fluorescence emission has been scaled.

FIG. 46A is a graph of overlapping spectra obtained from excitation scans of hybridization probe assays involving an acceptor probe labeled CY5.5 and a donor probe labeled FAM or DmF.

FIG. 46B is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probe assays involving an acceptor probe labeled with CY5.5 and a donor probe labeled with FAM or DmF.

FIG. 46C is a graph of overlapping spectra obtained from the emission scan shown at 46B, in which the fluorescence emission has been scaled.

FIG. 47A is a graph of overlapping spectra obtained from excitation scans of hybridization probe assays involving acceptor probes labeled with LC-Red 610 and donor probes labeled with FAM, Dmf, or Dam HEX.

FIG. 47B is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probe assays involving an acceptor probe labeled with LC-Red 610 and a donor probe labeled with FAM, DmF, or Dam HEX.

FIG. 48A is a graph of overlapping spectra obtained from excitation scans of hybridization probe assays involving acceptor probes labeled with CY3.5 and donor probes labeled with FAM, DmF, or Dam HEX.

FIG. 48B is a graph of overlapping spectra obtained from emission scans (absolute fluorescence) of hybridization probe assays involving an acceptor probe labeled with CY3.5 and a donor probe labeled with FAM, DmF, or Dam HEX.

Detailed Description

I. Definition of

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular biomolecules, methods, reaction mixtures, compositions, kits or systems, which can vary. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "biomolecule" includes a combination of two or more biomolecules. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology, and the grammatical variations thereof, will be used in accordance with the definitions set out below.

"5' -nuclease probe" refers to a labeled oligonucleotide that is capable of producing a detectable signal change when cleaved. For example, in certain embodiments, a 5' -nuclease probe comprises three labeled moieties, wherein one label emits radiation of increased intensity upon cleavage or other separation from the oligonucleotide. For example, in some of these embodiments, the 5 ' -nuclease probe is labeled at its 5 ' end with a quencher moiety, and near its 3 ' end with a pair of an acceptor moiety and a substantially non-fluorescent donor moiety. In certain embodiments, the 5' -nuclease probe is labeled at one or more positions other than, or in addition to, these terminal positions. When the probe is intact, energy transfer typically occurs between the label moieties such that the quencher moiety at least partially quenches the fluorescent emission from the acceptor moiety. In an extension step, e.g., a polymerase chain reaction, a 5 ' -nuclease probe bound to a template nucleic acid is cleaved by a 5 ' -3 ' nuclease activity, e.g., Taq polymerase, or by another polymerase having this activity, such that the fluorescent emission from the acceptor moiety is no longer quenched. In other exemplary embodiments, the 5' -nuclease probe comprises only an acceptor moiety and a substantially non-fluorescent donor moiety. When the moieties in these probes are cleaved apart from each other, a decrease in fluorescence emission from the acceptor moiety typically occurs. By way of further example, in certain embodiments, a 5' -nuclease probe comprises a self-complementary region such that the probe is capable of forming a hairpin structure under selected conditions. In these embodiments, the 5' -nuclease probe is also referred to herein as a "hairpin probe". Exemplary 5' -nuclease probes that can be adapted for use with the substantially non-fluorescent donor moieties described herein are also described, for example, in the following references: U.S. Pat. No. 5,210,015 to Gelfand et al, entitled "homology determination System for NUCLEASE Activity OF nucleic ACID POLYMERASE", 5.11.1993; U.S. Pat. No. 5,994,056 to Higuchi entitled "homologous METHODS FOR NUCLEIC acid amplification AND DETECTION", granted on month 11 AND 30 of 1999; AND Higuchi U.S. Pat. No. 6,171,785 entitled "METHODS AND DEVICES FOR NUCLEIC acids amplification AND detection of homologous NUCLEIC acids", granted on 1/9/2001.

"acceptor portion" or "acceptor" refers to a portion that is capable of accepting or absorbing energy transferred from an energy source. In some embodiments, the acceptor portion is also capable of emitting energy (e.g., light and/or heat) upon receiving a sufficient amount of the transferred energy. In these embodiments, the acceptor is also referred to as a "reporter moiety" or as a "reporter molecule". For example, certain acceptor moieties fluoresce in response to receiving a sufficient amount of non-fluorescent energy transferred from a substantially non-fluorescent donor moiety as described herein. Exemplary acceptor moieties include, but are not limited to, various fluorophores such as LightCyclers-Red 610(LC-Red 610), LC-Red 640, LC-Red 670, LC-Red 705, JA-270, CY5, CY5.5, etc.

"alkyl group" means a straight, branched or cyclic saturated hydrocarbon moiety, including all positional isomers, such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, octyl, nonyl, heptyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2-dimethylpropyl, 1-ethylpropyl, 1, 2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 2-trimethylpropyl, 1, 2, 2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-hexyl, cyclohexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl and n-decyl. The alkyl group typically contains from about 1 to about 20 carbon atoms, more typically from about 2 to about 15 carbon atoms. The alkyl group may be substituted or unsubstituted.

"amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. The term "amino acid" as used herein includes the following 20 naturally or genetically encoded alpha-amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The structure of these 20 natural amino acids is described in, for example, Stryer et al,Biochemistry5 th edition, Freeman Company (2002). Additional amino acids such as Selenocysteine and pyrrolysine may also be genetically encoded (Stadtman (1996) "seleniocysteine",Annu Rev Biochem.65: 83-100 and Ibba et al, (2002) "Genetic code: introducing pyrolysine (genetic code: introduction pyrrolysine) ",Curr Biol.12(13): R464-R466). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and/or backbones), and amino acid analogs. See, e.g., Zhang et al, (2004) "Selective incorporation of 5-hydroxytryptophan in-protein in mammalian cells" (Selective incorporation of 5-hydroxytryptophan into mammalian cell proteins) ",Proc.Natl.Acad.Sci.U.S.A.101(24): 8882-,Proc.Natl.Acad.Sci.U.S.A.101(20): 7566-7571, Ikeda et al, (2003) "Synthesis of a novel histidine analog and its incorporation into a protein in vivo",Protein Eng.Des.Sel.16(9): 699-,Science301(5635): 964-967, James et al, (2001) "Kinetic characterization of a photoisomerizable phenylazophenylalanine derivatives (a ribonuclease S mutant containing a photoisomerizable phenylazophenylalanine residue)",Protein Eng.Des.Sel.14(12): 983-: a general approach to the introduction of amber and ochre suppressor tRNA into mammalian cells: site-specific insertion of amino acid analogs into proteins) ",Proc.Natl.Acad.Sci.U.S.A.98(25): 14310-14315, Bacher et al, (2001) "Selection and Characterization of Escherichia coli Variants Cable of Growth on an aerobic tryptophan Analogue (Selection and Characterization of E.coli mutants Capable of growing on tryptophan analogues toxic to E.coli)",J.Bacteriol.183(18): 5414-5425, Hamano-Takaku et al, (2000) "A Mutant Escherichia coli Tyrosyl-tRNAS anythase acetyltyrosine kinase Moreeefficientlyth Tyrosine kinase (Mutant Escherichia coli Tyrosyl-tRNA synthetase Utilizes the Unnatural Amino Acid diazo Tyrosine more efficiently than Tyrosine)",J.Biol.Chem.275(51): 40324-,Protein Sci.10(7)：1281-1292。

by way of further example, an amino acid is typically an organic acid that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or side groups, or analogs of any of these groups. Exemplary side chains include, for example, mercapto, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, boronic acid, phosphoric acid, phosphonic acid, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or a combination of any of these groups. Other representative amino acids include, but are not limited to, amino acids comprising a photoactivatable cross-linking agent, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids capable of covalent or non-covalent interactions with other molecules, photocaged and/or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, carbon-linked sugar (carbon-linked sugar) -containing amino acids, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.

The term "amplification" in the context of a biomolecule refers to any process that results in an increase in the copy number of a biomolecule or a collection of biomolecules or their subsequences. As applied to nucleic acids, amplification refers to the generation of multiple copies of a polynucleotide or a portion of a polynucleotide, typically from a small number of polynucleotides (e.g., a single polynucleotide molecule), where the amplification product or amplicon is typically detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. Multiple copies of DNA are generated from one or a few copies of a target or template DNA molecule during Polymerase Chain Reaction (PCR) or Ligase Chain Reaction (LCR), which is a form of amplification. Amplification is not limited to the strict replication of the starting molecule. For example, RT-PCR is used to generate multiple cDNA molecules from a limited number of RNAs in a sample, in amplified form. In addition, multiple RNA molecules are produced from a single DNA molecule during transcription, which is also an amplified version.

The term "link" or "association" refers to the process of covalent and/or non-covalent association of two or more substances with each other, even if the process is only transient. For example, in certain embodiments, a substantially non-fluorescent donor moiety is attached to a compound as part of a method of producing a phosphoramidite. By way of further example, in some of the processes described herein, probe biomolecules bind to target biomolecules to effect detection of these targets.

"biomolecule" refers to an organic molecule produced and/or used by an organism and/or used to analyze an organism or component thereof. Exemplary biomolecules include nucleic acids, nucleotides, amino acids, polypeptides, peptides, peptide fragments, sugars, fatty acids, steroids, lipids, and combinations of these biomolecules (e.g., glycoproteins, ribonucleoproteins, or lipoproteins).

"biopolymer" refers to a biomolecule comprising at least two interconnected monomer units.

"biopolymer synthesis reagent" refers to a compound that can be used to synthesize a biopolymer or a component thereof. For example, in certain embodiments, the biopolymer synthesis reagent is a "nucleic acid synthesis reagent" such as a phosphoramidite or other reagent that can be used to synthesize an oligonucleotide or other nucleic acid. Exemplary oligonucleotide synthesis techniques optionally employed include the phosphoryl chloride (phosphonylchloride) method (Michelson et al, (1955)J.Chem.Soc.2632) Phosphodiester coupling method (Khorana et al, (1956)Chem.& Ind.London1523) Phosphotriester method (Letsinger et al, (1969)J.Am.Chem.Soc.91(12): 3360-J.Am.Chem.Soc.97: 3278-3279 and Letsinger et al, (1976)J.Am.Chem.Soc.98: 3655-3661) and the phosphoramidite method (Beaucage et al (1981)Tetrahedron Lett.22: 1859 1862 and McBride et al (1983)Tetrahedron Lett.24: 245-248). By way of further example, biopolymer synthesis reagents also include "polypeptide synthesis reagents" (e.g., t-Boc/Fmoc reagents) that can be used to synthesize peptides or other proteins. See for example Chan et al (editorial),Fmoc Solid Phase Peptide Synthesis：A Practical Approach Oxford Univthe quality Press (2000) and Jones,Amino Acid and Peptide Synthesis，oxford University Press (2002). Biopolymers can also be synthesized by many other methods known to those skilled in the art, such as by chemical ligation (see, e.g., Hackeng et al, (1999) "Protein synthesis by biological chemistry: Expanded scope by using direct methods for synthesis of proteins by natural chemistry)",PNAS 96(18)：10068-10073)。

"complement" in the context of nucleic acids refers to a nucleic acid or a segment thereof that can be combined with or hybridize to at least a subsequence of a nucleic acid in an antiparallel (antiparallelassification) manner. Antiparallel binding can be intramolecular, for example in the form of a hairpin loop within a nucleic acid, or intermolecular, for example when two or more single-stranded nucleic acids hybridize to one another. Certain bases not normally found in natural nucleic acids may be included in the nucleic acids referred to herein, including, for example, hypoxanthine, 7-deazaguanine, and the like. The complementarity need not be perfect; for example, a stable duplex or triplex may contain mismatched bases or unpaired bases. That is, where nucleic acids are only "partially complementary," antiparallel binding, whether intramolecular or intermolecular, can occur under certain conditions. One skilled in the art of nucleic acid chemistry can determine, for example, duplex or triplex stability by empirical judgment of a number of variables, such as the length of the region of complementarity, the base composition and sequence in the region of complementarity, ionic strength, melting temperature (T)_m) And the incidence of mismatched base pairs.

An "epitope" refers to a site on an antigen that is recognized and bound by an immunoglobulin or T cell receptor.

An "extendable nucleotide" refers to a nucleotide to which at least one other nucleotide can be added or covalently bonded once it is incorporated into a nucleotide polymer, for example, in a reaction catalyzed by a biocatalyst that catalyzes nucleotide incorporation. Examples of extendable nucleotides include deoxyribonucleotides and ribonucleotides, extendable nucleotides typically being extended by adding another nucleotide at the 3' position of their sugar moiety.

The term "extension" in the context of nucleic acids refers to the process by which one or more nucleotides are added or otherwise incorporated into a given nucleic acid.

An "extended primer nucleic acid" refers to a primer nucleic acid to which one or more additional nucleotides have been added or otherwise incorporated (e.g., covalently bonded).

"fluorescent dye" refers to a compound that is capable of absorbing energy from an energy source and emitting light in response to the absorbed energy. In some embodiments, certain fluorescent dyes function as acceptor moieties in the biomolecules described herein.

"hairpin probe" refers to a useful to achieve target nucleic acid detection oligonucleotides, including at least one self complementary region, the probe under selected conditions can form a hairpin or ring structure. Typically, hairpin probes include one or more labeling moieties. In an exemplary embodiment, the acceptor moiety and the substantially non-fluorescent donor moiety are positioned relative to each other in the hairpin probe such that energy transfer between the moieties occurs substantially only when the probe is in the hairpin conformation. In other embodiments, the hairpin probe includes a quencher moiety in addition to an acceptor moiety and a substantially non-fluorescent donor moiety. In some of these embodiments, the moieties are positioned in the probe such that the quencher moiety at least partially quenches an otherwise detectable signal generated by energy transfer between the acceptor moiety and the substantially non-fluorescent donor moiety when the probe is in the hairpin conformation. In contrast, when the probe in these embodiments is not in a hairpin conformation, the signal resulting from energy transfer between the acceptor moiety and the substantially non-fluorescent donor moiety is generally detectable. Thus, hairpin probes function similarly to molecular beacons in some of these embodiments. Hairpin probes may also serve as 5' -nuclease probes or hybridization probes in certain embodiments.

"hormone" refers to a biomolecule produced by an organism that produces a specific effect on the organism at a site other than the site of production of the organism. Hormones are usually produced by the endocrine system of multicellular organisms and generally exert either stimulatory or inhibitory effects on cellular activity. Hormones may also be synthesized in vitro.

"hybridization probe" refers to an oligonucleotide that includes at least one labeled moiety that can be used to effect detection of a target nucleic acid. In some embodiments, hybridization probes function in pairs. For example, in some of these embodiments, the first hybridization probe of the pair of hybridization probes includes at least one substantially non-fluorescent donor moiety at or near its 3 '-terminus, and the second hybridization probe includes at least one acceptor moiety (e.g., LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, JA-270, CY5, or CY5.5) at or near its 5' -terminus. The probes are typically designed such that when both probes hybridize to the target or template nucleic acid (e.g., during PCR), the first hybridization probe will bind to the 5-terminus or upstream of the second hybridization probe, bringing them into sufficient proximity for energy transfer to occur between the substantially non-fluorescent donor and acceptor moieties, thereby generating a detectable signal. Typically, the second hybridization probe also includes a phosphate or other group on its 3' -end to prevent it from extending during PCR. In certain embodiments, a hybridization probe pair comprises a substantially non-fluorescent donor moiety at or near the 5 '-terminus of one probe and an acceptor moiety at or near the 3' -terminus of the other probe. In another exemplary embodiment, one hybridization probe of a hybridization probe pair includes at least one substantially non-fluorescent donor moiety and at least one acceptor moiety, while the other hybridization probe includes at least one quencher moiety. In this embodiment, the moieties are positioned on both probes such that when both probes hybridize to the target nucleic acid, the quencher moiety will quench the fluorescence emitted from the acceptor moiety.

In the case of one polynucleotide to anotherIn the base pairing interaction of nucleotides (typically antiparallel polynucleotides), nucleic acids "hybridize" or "anneal" resulting in the formation of duplexes or other higher order structures commonly referred to as hybridization complexes. The primary interactions between antiparallel polynucleotides are typically base-specific interactions, such as A/T and G/C, that follow Watson/Crick and/or Hoogsteen-type interactions. It is not required that the two polynucleotides have 100% complementarity over their entire length to achieve hybridization. In some aspects, hybridization complexes may be formed from intramolecular interactions, or may be formed from intermolecular interactions. Hybridization occurs as a result of a variety of well characterized forces, including hydrogen bonding, solvent exclusion, and base stacking. An exhaustive guide to nucleic acid hybridization can be found in Tijssen,Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probeschapter 2 of section I, "Overview of the principles of hybridization and the strategy of nucleic acid probe assays", Elsevier (1993).

An "immunoglobulin" or "antibody" refers to a polypeptide substantially encoded by at least one immunoglobulin gene or at least one fragment of an immunoglobulin gene and involved in specific binding to a ligand. The term includes the natural form as well as fragments and derivatives. Fragments within the scope of this term as used herein include fragments produced by digestion with various peptidases, such as Fab, Fab ', and f (ab)' 2 fragments, fragments produced by chemical dissociation, fragments produced by chemical cleavage, and fragments designed recombinantly or otherwise artificially, so long as the fragment is still capable of specifically binding to the target molecule. Typical recombinant fragments, such as those produced by phage display, include single chain Fab fragments and scFv ("single chain variable") fragments. Derivatives within the scope of this term include antibodies (or fragments thereof) that have been modified in sequence, but are nevertheless capable of specifically binding to the target molecule, including interspecies chimeric and humanized antibodies. The antibodies or immunoglobulins used herein may be produced by any known technique, including harvest from natural B lymphocytes, hybridomas, recombinant expression systems, or phage-displayed cell cultures.

"linker moiety" or "linker" refers to a chemical moiety that is capable of covalently or non-covalently linking (e.g., ionically linking) a compound, a substituent, or a moiety to, for example, a solid support, another compound, another group, or another moiety. For example, the linker optionally connects a label (e.g., a substantially non-fluorescent donor moiety, an acceptor moiety) to the biomolecule. Linkers are generally bifunctional chemical moieties, and in certain embodiments they comprise a cleavable linkage that can be cleaved, for example, by heat, an enzyme, a chemical agent, and/or electromagnetic radiation, to release a substance or compound from, for example, a solid support or another compound. Careful selection of the linker allows cleavage to be performed under appropriate conditions to match the stability of the compound and assay. In general, a linker has no specific biological activity other than, for example, linking chemical moieties (e.g., substantially non-fluorescent donor and acceptor moieties) together or maintaining a certain minimum distance or other spatial relationship between the moieties. However, the composition of the linker may be selected to affect some property of the chemical moiety being attached, such as three-dimensional conformation, net charge, and/or hydrophobicity. Additional descriptions of linker molecules are found, for example, in Lyttle et al, (1996)Nucleic Acids Res.24(14): 2793, Shchepino et al (2001)Nucleosides，Nucleotides，& Nucleic Acids 20：369，Doronina et al(2001)Nucleosides，Nucleotides，& Nucleic Acids20: 1007, Tracwick et al (2001)Bioconjugate Chem.12: 900, Olejnik et al, (1998)Methods in Enzymology291: 135, Pljevaljcic et al, (2003)J.Am. Chem.Soc.125(12): 3486, Ward, et al, us 4,711,955, Stavrianopoulos, us 4,707,352 and Stavrianopoulos, us 4,707,440.

"lipid" refers to a water-insoluble biomolecule comprising fatty acids, sterols, and/or isoprenoid compounds. Exemplary lipid groups include fatty acids (e.g., oleic acid, palmitic acid, and stearic acid)) Neutral fats (e.g., coconut oil and tallow), phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol), sphingolipids (e.g., sphingomyelin), sugar esters (e.g., cerebrosides and gangliosides), steroids (e.g., cholesterol), and terpenes (e.g., essential oils and carotenoids). In the context of for example the Vance et al,Biochemistry of Lipids，Lipoproteins and Membranes，lipids are also described in Elsevier Science (2002), 4 th edition.

"lipid mixing assay" refers to an assay that involves detecting one or more properties of lipids when they are combined or pooled with each other. For example, some of these assays involve fusing together individual membranes comprising individual label moieties such as substantially non-fluorescent donor moieties, acceptor moieties, and/or quencher moieties as described herein. Lipid mixing assays are also described, for example, in the following documents: blumenhal et al, (2002) "Fluorescent lipid probes in the study of viral membrane fusion",Chem Phys Lipids116: 39-55, Hoekstra et al, (1993) "Lipid mixing assays for determining fusion in liposome systems",Methods Enzymol220: 15-32, Hoekstra (1990) "Fluorescence assays to monitor membrane fusion: potential application of the potential in biliary lipid secretion and vesicle interactions to monitor membrane fusion ",Hepatology12: 61S-66S, Stegmann et al, (1989) "Protein-mediated membrane fusion",Annu Rev Biophys Biophys Chem18: 187- "Fluorescence method for measuring the kinetics of fusion between biological membranes", Hoekstra et al, (1984) "Fluorescence method for measuring the fusion between biological membranes",Biochemistry23: 5675-"en (en)"),Biochemistry20: 4093-,Biochemistry 32：11330-11337。

"monomer unit" or "monomer" refers to a chemical compound capable of undergoing polymerization. Exemplary monomer units include nucleotides that can polymerize to form nucleic acids, amino acids that can polymerize to form proteins, and monosaccharides that can polymerize to form polysaccharides.

"mixture" refers to a combination of two or more different ingredients. "reaction mixture" refers to a mixture comprising molecules that can participate in and/or facilitate a given reaction or assay. For example, an amplification reaction mixture typically includes a solution containing the reagents required to perform the amplification reaction, and typically contains primers, a biocatalyst that catalyzes nucleotide incorporation, dntps, and a divalent metal cation in a suitable buffer. A reaction mixture is referred to as a complete reaction mixture if it contains all the reagents required to carry out the reaction, and as an incomplete reaction mixture if it contains only a subset of the required reagents. Those skilled in the art will recognize that reaction components are conventionally stored as separate solutions, each solution containing a subset of the total components for convenience, storage stability, or to allow for adjustment of component concentrations depending on the application, and that the reaction components may be combined prior to reaction to produce a complete reaction mixture. Furthermore, one skilled in the art will recognize that each reaction component is commercially available in separate packages, and that useful commercially available kits may contain any subset of each reaction or assay component, including the biomolecules of the invention.

"moiety" or "group" refers to one of the various portions (e.g., functional groups or substituents) into which a substance, such as a molecule, is divided. For example, a biomolecule of the invention includes at least one substantially non-fluorescent donor moiety.

The term "monitoring" in the context of a given process, reaction or assay refers to the periodic or continuous observation, detection, testing and/or quantification of one or more aspects or properties of the process, reaction or assay or component thereof (component). For example, in certain PCR-based assays, the intensity of light emitted from the receiver portion is periodically or continuously detected during each reaction cycle. The monitoring process is typically at least partially automated.

A "non-fluorescent donor moiety" refers to a moiety that is capable of transferring, emitting, or donating one or more forms of excitation energy to one or more acceptor moieties, but which is not a detectable fluorescent form of energy. "substantially non-fluorescent donor moiety" refers to a moiety that is capable of transferring, emitting, or donating one or more forms of excitation energy to one or more acceptor moieties, but that emits substantially or little detectable fluorescence from the donor. For example, the ratio of detectable absolute fluorescent emission from the former to detectable absolute fluorescent emission from the latter is typically about 500: 1 or greater, more typically about 1000: 1 or greater, and even more typically about 1500: 1 or greater (e.g., about 2000: 1, about 2500: 1, about 3000: 1, about 3500: 1, about 4000: 1, about 4500: 1, or about 5000: 1) at substantially the same concentration of the fluorescent moiety and the substantially non-fluorescent donor moiety. Exemplary substantially non-fluorescent donor moieties include 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorescein, and 5-carboxy-amino pentachlorofluorescein. The non-fluorescent donor moiety and the substantially non-fluorescent donor moiety will typically respond by emitting non-fluorescent excitation energy upon absorption of a sufficient amount of energy from another energy source (e.g., a light source and/or a heat source). In addition, acceptor moieties are typically capable of absorbing excitation energy emitted from these donor moieties and in response, emitting fluorescence.

The term "non-fluorescent energy" refers to energy other than that emitted in the form of fluorescence.

The term "nucleic acid" or "polynucleotide" refers to a polymer that can be converted to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. This includes polymers of nucleotides such as RNA and DNA and their modified forms, Peptide Nucleic Acids (PNA) and Locked Nucleic Acids (LNA)^TM). In certain embodiments, the nucleic acid can be a polymer comprising multiple monomer types, e.g., both RNA and DNA subunits. The nucleic acid can be or can include, for example, a chromosome or chromosome segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, a product of a Polymerase Chain Reaction (PCR), an oligonucleotide, a probe, and a primer. The nucleic acid may be, for example, a single-stranded nucleic acid, a double-stranded nucleic acid, or a triple-stranded nucleic acid, and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence optionally comprises or encodes a complementary sequence in addition to any specifically indicated sequence.

Nucleic acids are not limited to molecules having a native polynucleotide sequence or structure, a native backbone, and/or native internucleotide linkages. For example, nucleic acids containing one or more carbocyclic sugars are also included within this definition (Jenkins et al (1995)Chem.Soc.Rev.Pages 169-176). By way of further example, nucleic acids, while typically containing phosphodiester linkages, also include nucleic acid analogs having alternative (alternate) backbones in some cases. These backbones include, but are not limited to, the phosphoramide linkage (Beaucage et al, (1993)Tetrahedron49(10): 1925 and references therein; letsinger (1970)J.Org.Chem.35: 3800; sprinzl et al, (1977)Eur.J. Biochem.81: 579; letsinger et al (1986)Nucl.Acids Res.14: 3487; sawai et al (1984)Chem.Lett.805; letsinger et al (1988)J.Am.Chem.Soc.110: 4470 and Pauwels et al (1986)Chemica Scripta26: 1419) phosphorothioate linkages (Mag et al, (1991)Nucleic Acids Res.19: 1437 and U.S. Pat. No. 5,644,048), dithiophosphate linkage (Briu et al, (1989)J.Am.Chem.Soc.111: 2321) o-methyl phosphoramidate (phophoroamidate) linkages (Eckstein,Oligonucleotides and Analogues：A Practical Approachoxford university Press (1992)) and peptide nucleic acid backbones and linkages (Egholm (1992)J.Am. Chem.Soc.114: 1895; meier et al (1992)Chem.Int.Ed.Engl.31：1008；Nielsen(1993)Nature365: 566 and Carlsson et al, (1996)Nature380: 207). Other similar nucleic acids include those having a positively charged backbone (Denpcy et al, (1995)Proc. Natl.Acad.Sci.USA92: 6097) nucleic acids having a nonionic backbone (U.S. Pat. nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; angew (1991)Chem.Intl.Ed.English30: 423; letsinger et al (1988)J.Am.Chem.Soc.110: 4470; letsinger et al (1994)Nucleoside & Nucleotide13: 1597; ASCSymposium Series 580, "Carbohydrate Modifications in antisense research", Y.S. Sanghvi and P.Dan Cook (eds.), chapters 2 and 3; mesmaeker et al (1994)Bioorganic & Medicinal Chem.Lett.4: 395; jeffs et al (1994)J.Biomolecular NMR 34：17；Tetrahedron Lett.37: 743(1996)) and nucleic acids having a non-ribose backbone, including those having a non-ribose backbone described in U.S. Pat. Nos. 5,235,033 and 5,034,506 and ASC Symposium Series 580, carbohydrate reagents in Antisense Research, Y.S. Sanghvi and P.Dan Cook (eds.), chapters 6 and 7. In the case of e.g. Rawls,C & E Newsseveral nucleic acid analogs are also described on page 35, 2.6.1997. Modifications of the ribose-phosphate backbone may be made to facilitate the addition of additional moieties, such as labeling moieties, or to alter the stability and half-life of such molecules in physiological environments.

In addition to the natural heterocyclic bases (e.g., adenine, guanine, thymine, cytosine, and uracil) that are typically found in nucleic acids, nucleic acid analogs also include nucleic acids having non-natural heterocyclic bases or other modified bases. For example, some are used in nucleotides to act as melting temperatures (T)_m) The base of the modifying agent is optionally included. For example, some of these bases include 7-deazapurines (e.g., 7-deazapurine or 7-deazapurine), pyrazolo [3, 4-d]Pyrimidines and propynyl groups-dN (e.g. propynyl-dU or propynyl-dC). See, for example, Seela, U.S. Pat. No. 5, 5,990,303, entitled "SYNTHESIS OF 7-DEACA-2 '-DEOXYGUANOSINUCLEOTIDES (Synthesis of 7-DEAZA-2' -deoxyguanosine nucleotide)", granted 11/23 of 1999. Other representative heterocyclic bases include, for example, hypoxanthine, xanthine; 8-aza derivatives of 2-aminopurine, 2, 6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine and xanthine; 7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine, 2, 6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynyl cytosine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil; 5-trifluoromethyl uracil; 5-methoxymethyluracil; 5-ethynyluracil and 5-propynyluracil. In, for example, Seela et al (1991)Helv. Chim.Acta74: 1790. grein et al (1994)Bioorg.Med.Chem.Lett.4: 971-Helv.Chim.Acta82: 1640.

Additional examples of modified bases and nucleotides are also described, for example, in the following documents: froehler et al, U.S. Pat. No. 5, 5,484,908 entitled "oligonucleotide 5-PROPYNYL PYRIMIDINES", granted 1/16/1999; froehler et al, U.S. Pat. No. 3, 5,645,985, entitled "ENHANCED TRIPLE-HELIX AND DOUBLE-HELIXFORMATION WITH OLIGOMERS CONTAINING MODIFIEDPYRIMIDINES (enhanced triple AND DOUBLE HELIX structure WITH modified pyrimidine-CONTAINING OLIGOMERS)", issued on 7/8 of 1997; froehler et al, U.S. Pat. No. 5, 5,830,653 entitled "METHODS OF USING OLIGOMERS CONTAINING modified pyrimidines", granted on 3/11/1998; kochkine et al, U.S. Pat. No. 6,639,059 entitled "SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES ([2.2.1] Synthesis of bicyclic NUCLEOSIDES)", granted on month 10 and 28 of 2003; skoov U.S. Pat. No. 6,303,315 entitled "ONE STEP AMPLE PREPARATION AND DETECTION OF NUCLEIC ACID COMPOUND BIOLOGICAL SAMPLES (ONE-step sample PREPARATION AND DETECTION OF NUCLEIC acids in COMPLEX BIOLOGICAL SAMPLES)," 10/16/2001, U.S. Pat. App. Pub. No. 2003/0092905 entitled "SYNTHESIS OF [2.2.1] BICYCLONLEOSIDES ([2.2.1] Synthesis OF bicyclic nucleosides)", published 5/15/2003.

"nucleoside" refers to a nucleic acid component comprising a base or basic group (e.g., comprising at least one carbocyclic ring (carbocyclic ring), at least one heterocyclic ring and/or at least one aryl group) covalently linked to a sugar moiety (e.g., ribose), a derivative of a sugar moiety or a functional equivalent of a sugar moiety (e.g., an analog such as a carbocyclic ring). For example, when a nucleoside includes a sugar moiety, the base is typically attached to the 1' -position of the sugar moiety. As previously mentioned, the base may be a natural base (e.g. a purine base such as adenine (A) or guanine (G), a pyrimidine base such as thymine (T), cytosine (C) or uracil (U)), or a non-natural base (e.g. a 7-deazapurine base, a pyrazolo [3, 4-d ] pyrimidine base or an alkynyl-dN base). Exemplary nucleosides include ribonucleosides, deoxyribonucleosides, dideoxyribonucleosides, and carbocyclic nucleosides.

"nucleotide" refers to an ester of a nucleoside, for example, a phosphate ester of a nucleoside. For example, a nucleotide can include 1, 2, 3, or more phosphate groups covalently attached to the 5' position of the nucleoside sugar moiety.

"biocatalyst that catalyzes nucleotide incorporation" refers to a catalyst that is capable of catalyzing the incorporation of nucleotides into nucleic acids. The biocatalyst that catalyzes the nucleotide incorporation is typically an enzyme. An "enzyme" is a protein and/or nucleic acid based catalyst that acts to reduce the activation energy of a chemical reaction involving another compound or "substrate". "an enzyme that catalyzes nucleotide incorporation" refers to an enzyme that catalyzes the incorporation of nucleotides into nucleic acids, for example, in a nucleic acid amplification process. Exemplary enzymes that catalyze nucleotide incorporation include, for example, polymerases, terminal transferases, reverse transcriptases, telomerase, polynucleotide phosphorylases, and ligases. "thermostable enzyme" refers to an enzyme that is stable to heat, resists heat, and retains sufficient catalytic activity when subjected to elevated temperatures for a selected period of time. For example, a thermal temperature polymerase enzyme, when subjected to high temperatures for the time required to effect denaturation of double-stranded nucleic acids and at temperatures that allow hybridization of the primers, still retains sufficient activity to effect a subsequent primer extension reaction. The heating conditions required for nucleic acid denaturation are well known to those skilled in the art and are exemplified in the following patents: mullis, U.S. Pat. No. 4,683,202, entitled "PROCESS FOR AMPLIFYING NUCLEIC ACID SEQUENCES (method FOR AMPLIFYING NUCLEIC ACID SEQUENCES)", issued 28.7.198, and Mullis et al, U.S. Pat. No. 4,683,195, entitled "PROCESS FOR AMPLIFYING, DETECTING, AND/OR-CLONING NUCLEIC ACID SEQUENCES (method FOR AMPLIFYING, detecting and/OR CLONING NUCLEIC ACID SEQUENCES)", issued 28.7.7.198. By way of further example, "thermostable polymerases" refers to enzymes suitable for temperature cycling reactions, such as the polymerase chain reaction ("PCR"). For thermostable polymerases, enzymatic activity refers to catalyzing the combination of nucleotides in an appropriate manner to form a primer extension product that is complementary to a template nucleic acid.

"oligonucleotide" refers to a nucleic acid that includes at least two monomeric units (e.g., nucleotides) of a nucleic acid, typically includes more than three monomeric units, and more typically includes more than ten monomeric units. The exact size of the oligonucleotide will generally depend on a number of factors, including the ultimate function or use of the oligonucleotide. Typically, nucleoside monomers are linked by phosphodiester linkages or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroamidate, and methyl phosphonate, if associated with a counter ion such as H⁺、NH₄ ⁺And Na⁺These counter ions are also included if present. Oligonucleotides are optionally prepared by any suitable method including, but not limited to, isolation of existing or native sequences, DNA replication or amplification, reverse transcription, cloning of appropriate sequences and restriction digestion, or by direct chemical synthesis such as: phosphotriester method, Narang et al, (1979)Meth.Enzymol.68: 90-99; phosphodiester method, Brown et al, (1979)Meth. Enzymol.68: 109-; diethyl phosphoramidite method, Beaucage et al, (1981)Tetrahedron Lett.22: 1859-; triester method, Matteucci et al, (1981)J.Am. Chem.Soc.103: 3185 and 3191; an automatic synthesis method; or solid phase hybrid method, U.S. Pat. No. 4,458,066 to Caruthers et al, entitled "PROCESS FOR PREPARINGPOLYNEUCLEOTIDES (method FOR preparing polynucleotides)", granted 7/3/1984; or other methods known to those skilled in the art.

The term "peak visible absorption" refers to the wavelength at which a molecule is most effective at absorbing energy in the visible or optical region of the electromagnetic spectrum. The visible or optical region of the electromagnetic spectrum typically includes wavelengths in the range from about 700 nanometers (nm) to about 400 nm. For example, in some embodiments, non-fluorescent energy transfer from a substantially non-fluorescent donor moiety to an acceptor moiety is most effective when the peak visible absorptions of the substantially non-fluorescent donor moiety and the acceptor moiety differ by about 100nm or more. Depending on the particular molecule being studied, the peak visible absorption can correspond to the overall maximum absorbance (global absorbance maximum) or local maximum absorbance (local absorbance maximum) of the molecule in the visible region of the electromagnetic spectrum.

"phosphoramidite" is meant to include compounds comprising a group of the formula:

wherein R is₁And R₂Is an alkyl group independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and heptyl; r₃Is (CH)₂)₂CN or CH₃. For example, in certain embodiments, phosphoramidites are nucleoside-3' -ylidenes commonly used in oligonucleotide synthesis proceduresA phosphoramide monomer. In some of these embodiments, the phosphoramidite monomers are protected at the 5' hydroxyl position with a protecting group. Usually, different protecting groups are also attached to the exocyclic amine of the base. In addition, the phosphorus atom of the monomer is optionally substituted by a beta-cyanoethyl (R3) group and diisopropylamine (NR) group corresponding to the above-shown chemical formula₁R₂) Groups or other group protection. Phosphoramidites and oligonucleotide synthesis is described, for example, in Beaucage et al, (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite approach",Tetrahedron48: 2223-2311 are also described.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids. These terms apply to amino acid polymerases in which one or more amino acid residues are an analogue, derivative or mimetic of the corresponding natural amino acid, and to natural amino acid polymers. For example, polypeptides may be modified or derivatized, e.g., by the addition of carbohydrate residues to form glycoproteins, by the addition of lipid moieties to form lipoproteins. Thus, the terms "polypeptide", "peptide" and "protein" include glycoproteins and lipoproteins as well as non-glycoproteins and non-lipoproteins.

A "primer nucleic acid" or "primer" is a nucleic acid that hybridizes to a target or template nucleic acid and allows strand extension or elongation to occur under appropriate reaction conditions using, for example, a biocatalyst that catalyzes nucleotide incorporation, such as a polymerase. The primer nucleic acid is typically a natural or synthetic oligonucleotide (e.g., a single stranded oligodeoxynucleotide). Primer nucleic acids typically comprise a hybridizing region in the range of about 8 to about 100 nucleotides in length, although other primer nucleic acid lengths are optionally employed. Short primer nucleic acids generally require cooler temperatures to form sufficiently stable hybridization complexes with the template nucleic acid. Primer nucleic acids that are at least partially complementary to subsequences of the template nucleic acid are generally sufficient to hybridize to the template and allow extension to occur. Primer nucleic acids can be labeled, if desired, by incorporating a label that can be detected, for example, by spectroscopic, photochemical, biochemical, immunological, chemical, or other techniques. For example, useful labels include substantially non-fluorescent donor moieties, acceptor moieties, quencher moieties, radioisotopes, electron-dense reagents (electron-densenerates), enzymes (commonly used to perform ELISA), biotin or haptens and proteins for which antisera or monoclonal antibodies are available. Many of these and other markers are further described herein and/or otherwise known in the art. One skilled in the art will recognize that in certain embodiments, primer nucleic acids may also be used as probe nucleic acids.

"Probe biomolecule" refers to a labeled or unlabeled biomolecule that is capable of selectively binding to a target biomolecule.

"protecting group" refers to a chemical group that is covalently or non-covalently bonded to a given compound to prevent unwanted chemical reactions at one or more sites in the compound. Exemplary protecting groups include trityl, monomethoxytrityl, dimethoxytrityl, Jerusalem artichoke glycosyl, fluorenylmethoxycarbonyl and benzhydryloxycarbonyl.

The term "probe nucleic acid" or "probe" refers to a labeled or unlabeled oligonucleotide capable of selectively hybridizing to a target or template nucleic acid under suitable conditions. Typically, the probe is sufficient to be complementary to a particular target sequence contained in the nucleic acid sample to form a stable hybridization duplex with that target sequence under selected hybridization conditions, such as, but not limited to, stringent hybridization conditions. Hybridization assays using probes under sufficiently stringent hybridization conditions allow for selective detection of specific target sequences. The term "hybridizing region" refers to that region of a nucleic acid which is fully or substantially complementary to a target sequence, and thus capable of hybridizing to the target sequence. To distinguish single nucleotide differences in sequences in hybridization assays, the length of the hybridization region is typically from about 8 to about 100 nucleotides. While the hybridization region generally refers to the entire oligonucleotide, the probe may also include additional nucleotide sequences that serve, for example, as linker binding sites (linkerbinding sites) to provide sites to attach the probe sequences to a solid support. Probes of the invention are generally included in compositions comprising one or moreA labeled (e.g., substantially non-fluorescent donor, acceptor, and/or quencher moiety) nucleic acid, such as a 5' -nuclease probe, hybridization probe, Fluorescence Resonance Energy Transfer (FRET) probe, hairpin probe, or molecular beacon, can also be used to detect hybridization between the probe and target nucleic acid in the sample. In some embodiments, the hybridization region of the probe is fully complementary to the target sequence. In general, however, complete complementarity is not necessary (i.e., the nucleic acids may be partially complementary to each other); a stable hybridization complex may contain mismatched bases or unpaired bases. Modifications to stringent conditions may be required to allow stable hybridization complexes with one or more base pair mismatches or unpaired bases. The contents of Sambrook et al,Molecular Cloning：A Laboratory Manualguidance regarding appropriate modifications is provided in 3 rd edition, Cold Spring Harbor Laboratory Press, Gold Spring Harbor, N.Y. (2001). The stability of the target/probe hybridization complex depends on a number of variables, including the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, temperature, and ionic conditions. One skilled in the art will recognize that, in general, the correct complementary sequence (complement) for a given probe may also be used as a probe. One of skill in the art will also recognize that in certain embodiments, probe nucleic acids may also be used as primer nucleic acids.

The term "proximity assay" refers to an assay in which the generation of a detectable signal depends, at least in part, on the acceptor moiety and donor moiety being sufficiently close to each other. For example, in certain embodiments, each biomolecule comprises an acceptor moiety and a donor moiety, respectively, such that the generation of a detectable energy emission from the acceptor moiety is at least partially dependent on the intermolecular distance of each biomolecule. In other exemplary embodiments, where each biomolecule comprises both an acceptor moiety and a donor moiety (e.g., the same or different monomeric units attached to the biopolymer), the generation of detectable energy emissions from the acceptor moiety is at least partially dependent on the intramolecular spacing between the moieties. In some of these embodiments, the detectable energy emission from the acceptor moiety will change, for example, as the biomolecule undergoes a conformational change, or as the acceptor moiety and donor moiety are separated from each other in the biomolecule by an enzymatic process or other cleavage process.

A "quencher moiety" or "quencher" refers to a moiety that is capable of reducing the detectable emission of radiation (e.g., fluorescent or luminescent radiation) from a radiation source that would otherwise emit such radiation. Quenchers typically reduce the detectable radiation emitted by the radiation source by at least 50%, typically by at least 80%, more typically by at least 90%. Certain quenchers can re-emit energy absorbed from, for example, a fluorescent dye in the form of their characteristic signal, and thus the quenchers can also be acceptor moieties. This phenomenon is commonly referred to as fluorescence resonance energy transfer or FRET. Alternatively, the quencher may emit the energy absorbed from the fluorescent dye in a form other than light, such as heat. Common molecules used in FRET applications include, for example, fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL, and EDANS. Whether a fluorescent dye is an acceptor or a quencher is determined by its excitation and emission spectra and the fluorescent dye with which it is paired. For example, FAM is most efficiently excited by light having a wavelength of 494nm and emits light in the spectral range of 500-650nm with a maximum emission wavelength of 525 nm. FAM is a donor moiety suitable for use, for example, with TAMRA as a quencher, with a maximum excitation wavelength of 560 nm. Exemplary non-fluorescent Quenchers or dark Quenchers capable of dissipating energy absorbed from fluorescent dyes include Black Hole Quenchers sold by Biosearch technologies, Inc. (Novato, Calif., USA)^TM。Black Hole Quenchers^TMIs a structure comprising at least three radicals (radial) selected from substituted or unsubstituted aryl or heteroaryl compounds or combinations thereof, wherein at least two of these residues are linked by an exocyclic diazo linkage (see, e.g., international publication WO 01/86001 to Cook et al, titled "DARK quers ford-ACCEPTOR-accepter ENERGY TRANSFER (DARK quencher for donor-ACCEPTOR energy transfer)", published 11/15/2001). Exemplary quenchers are also provided, for example, in U.S. Pat. No. 6,465,175 to Horn et al, entitled "OLIGONUCLEOTIDEPROBES BEARING QUENCABLE FLUORESCENT LABELS, and methods OF using THEREOF (withQuenchable fluorescent-labeled oligonucleotide probes and methods of use) "granted on days 10/15 in 2002.

The "sequence" of a biopolymer refers to the order and identity (identity) of the monomeric units (e.g., nucleotides, amino acids, and monosaccharides) in the biopolymer. Nucleic acid sequences are usually from 5 '-3' direction reading, and polypeptide sequences are usually from the amino terminal or N terminal to the carboxyl terminal or C terminal direction reading.

"solid support" refers to a solid material that can be derivatized with or otherwise attached to a chemical moiety, such as a probe. Exemplary solid supports include plates (plates), beads (beads), microbeads (microbeads), Controlled Pore Glass (CPG), polystyrene, tubes, fibers, whiskers (whisker), combs, hybridization chips (including microarray substrates such as GeneChip)Probe arrays (substrates used in Affymetrix, inc., Santa Clara, CA, USA), films, single crystals, ceramic layers, self-assembled monolayers, and metal surfaces.

The term "stringent" or "stringent conditions" as used herein means hybridization conditions of low ionic strength and high temperature, which are well known in the art. See for example, Sambrook et al,Molecular Cloning： A Laboratory Manual3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001);Current Protocols in Molecular Biology(Ausubel et al (eds.), J.Wiley&Sons inc, New York, 1997); tijssen (1993), supra. Generally, stringent conditions are selected to match the thermal melting point (T) of the designated sequence at a defined ionic strength and pH_m) About 5-30 deg.c lower. Alternatively, stringent conditions are selected to compare the T of a given sequence at a defined ionic strength and pH_mAbout 5-15 deg.c lower. T is_mProbes that are complementary to the target sequence are 50% in hybridization equilibrium with the target sequence (at T due to the excess presence of the target sequence_mThe lower 50% of the probes are in hybridization equilibrium) at a temperature determinedIonic strength, pH and nucleic acid concentration).

"subsequence", "segment" or "fragment" refers to any portion (portion) of the entire biopolymer sequence.

The term "sufficiently close" in the context of energy transfer between a donor moiety and a acceptor moiety refers to the appropriate spacing and/or positioning of the moieties relative to one another such that the acceptor moiety is capable of accepting or absorbing energy transferred from the donor moiety. For example, certain acceptor moieties fluoresce in response to receiving a sufficient amount of transferred non-fluorescent energy from a spaced and/or positioned suitable substantially non-fluorescent donor moiety.

"sugar" or "carbohydrate" refers to a polyhydroxyaldehyde (aldose) or polyhydroxyketone (ketose), or a compound or analog derived therefrom. Carbohydrate polymers (e.g., oligosaccharides and polysaccharides) include monosaccharide units. Exemplary carbohydrates include glyceraldehyde, erythrose (erthyrose), threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, dihydroxyacetone, xylulose, and fructose. Sugar analogs such as carbocyclic sugars are also included in the definition of sugars (see, e.g., Jenkins et al (1995)Chem.Soc.Rev.Pages 169-176).

"System" refers to a set of objects and/or devices that form a network to accomplish a desired goal. For example, in some embodiments, the systems of the invention comprise a container and/or solid support comprising one or more biomolecules comprising a substantially non-fluorescent donor moiety and a detection component configured to detect light emitted from the acceptor moiety when the acceptor moiety is sufficiently proximal to the substantially non-fluorescent donor moiety, e.g., as part of a hybridization probe assay.

"target" refers to a biomolecule or portion thereof (a portion) to be amplified, detected, and/or otherwise analyzed.

A "terminator nucleotide" refers to a nucleotide that, when incorporated into a nucleic acid, prevents further extension of the nucleic acid by, for example, at least one biocatalyst that catalyzes nucleotide incorporation.

When thermal energy is or can be transferred between objects, "heat transfer" between the objects occurs. For example, in certain embodiments of the systems described herein, the thermal modulator is in thermal communication with the vessel and/or the solid support to modulate the temperature in the vessel and/or on the solid support.

Introduction II

The present invention relates generally to the transfer of non-fluorescent energy between a donor moiety and a acceptor moiety. More specifically, the substantially non-fluorescent donor moieties described herein can be used in almost any application involving energy transfer between a donor moiety and a acceptor moiety, including many different proximity assay formats. For example, conventional energy transfer dye pairs are typically configured such that the short wavelength fluorescent donor moiety transmits absorbed energy to the longer wavelength acceptor moiety. Furthermore, it is generally believed that the fluorescence emission spectrum of the donor moiety and the fluorescence absorption spectrum of the acceptor moiety must overlap. Contrary to this general teaching, one unexpected result of the present invention is that the donor moieties described herein lack a measurable (apremicable) fluorescence emission spectrum. However, when the acceptor moieties are positioned sufficiently close to the donor moieties, the acceptor moieties will emit light in response to absorbing the non-fluorescent energy transferred from the donor moieties.

A common problem with the use of conventional fluorescent donor moieties such as fluorescein is the background fluorescence emitted by the donor moiety itself. This background fluorescence can make it difficult to discern the energy transfer to longer wavelength acceptor moieties, particularly in multiplex applications involving the use of multiple fluorescent donor probes. In particular, background or baseline light emissions such as these often negatively impact the performance of the assay by, for example, limiting the sensitivity of the assay, i.e., the ability of the assay to distinguish small differences in analyte concentration, and the dynamic range of the assay, i.e., the useful range of the assay extending from the lowest concentration at which quantitative measurements can be made (limit of quantitation or LOQ) to the concentration at which the calibration curve deviates from linearity (limit of linearity, LOL). Thus, there are a number of advantages to using substantially non-fluorescent donor moieties as described herein, one of which is that this type of background fluorescence is reduced or eliminated, thereby improving the performance of assays involving these types of donor moieties. The improved signal-to-noise ratio provided by the donor moieties described herein, along with many other features of the present invention, are further illustrated in the examples provided below.

The present invention provides biomolecules (e.g., biopolymer synthesis reagents, oligonucleotides, polypeptides, carbohydrates, and lipids) comprising a substantially non-fluorescent donor moiety as described herein covalently or otherwise attached thereto, as described in more detail below. The present invention also provides various reaction mixtures comprising these biomolecules, along with other reagents to perform a given process, such as biopolymer synthesis, biopolymer labeling, nucleic acid detection, in addition to many other processes described herein or otherwise known to those skilled in the art. The present invention also provides various methods, which are also described herein. For example, in certain embodiments, the present invention provides methods of performing proximity assays and methods of detecting target biomolecules. For example, the biomolecules described herein are optionally used in methods for detecting nucleic acids in a variety of applications, including genotyping, diagnostics, forensic and many other applications well known to those skilled in the art. Further by way of example, methods of sequencing and/or labeling biopolymers, and methods of synthesizing the biomolecules described herein are also provided. The present invention further provides certain commercial processes. In addition, the invention also provides various kits and systems. These and various other aspects and features of the present invention will become apparent after review of the present specification.

Substantially non-fluorescent donor moieties

Almost any compound that is capable of transferring non-fluorescent energy to an acceptor moiety without emitting measurable fluorescence during the process can be used as the substantially non-fluorescent donor moiety. Thus, there is no attempt to list all possible compounds that may optionally be employed for this purpose. Nevertheless, certain specific substantially non-fluorescent donor moieties are mentioned herein to further illustrate aspects of the invention. Specifically, representative substantially non-fluorescent donor moieties or precursors thereof include 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-2 ', 4, 4', 5 ', 7, 7' -hexachlorofluorescein, 6-carboxy-amino-pentachlorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluorofluor, 5-carboxy-2 ', 4, 4', 5 ', 7, 7' -hexachloro. The chemical structures of some of these exemplary moieties are shown in table I.

TABLE I

Note that under certain conditions (e.g., when HEX-labeled oligonucleotides are heated in ammonia at 55 ℃ for about 24 hours), attachment of 6-carboxy-2 ', 4, 4', 5 ', 7, 7' -hexachlorofluorescein and 5-carboxy-2 ', 4, 4', 5 ', 7, 7' -hexachlorofluorescein to a biomolecule is achieved by loss of at least one chloride ion and addition of at least one amino group, and thus, when attached to a biomolecule in these embodiments, these moieties are referred to herein as "6-carboxy-amino pentachlorofluorescein" and "5-carboxy-amino pentachlorofluorescein", respectively, or as "damaged HEX" or "Dam HEX" (e.g., 5-Dam HEX or 6-Dam HEX). Furthermore, for all molecular structures provided herein, it is specified that these molecular structures not only encompass the exact electronic structures given, but also all resonance structures and protonation states thereof.

Many substantially non-fluorescent donor moieties can be readily synthesized using chemical methods well known to those skilled in the art. Various synthetic techniques optionally applicable to these synthetic schemes are well known to those skilled in the art and are also described, for example, in the following references: the mark is a mark of the mark, and the mark is a mark of the mark,Advanced Organic chemistry: reactions, Mechanisms, and structures5 th edition, John Wiley&Sons, Inc. (2000), Carey and Sundberg,Advanced Organic Chemistry，Part a: structure and Mechanism4 th edition, Plenum Publishing Corporation (2000), and Carey and Sundberg,Advanced Organic Chemistry，Part B： Reaction and Synthesis4 th edition, Plenum Publishing Corporation (2001). Chemical starting materials and other reaction components useful for the synthesis of the substantially non-fluorescent donor moiety are readily available from a number of commercial suppliers, including, for example, -Aldrich, inc. (St Louis, MO, USA). In addition, certain substantially non-fluorescent donor moieties are pre-synthesized and available from various commercial suppliers such as Molecular Probes, inc. (Eugene, OR, USA).

Reagents comprising a substantially non-fluorescent donor moiety

The invention also relates to reagents comprising substantially non-fluorescent donor moieties as described herein. These reagents may include any molecule or material to which these donor moieties can be covalently or non-covalently attached. Examples of such molecules and materials include, but are not limited to, solid supports (e.g., membranes or controlled pore glass), viral particles, tissues, cells (e.g., mammalian cells and bacteria or other microorganisms), cellular organelles, and organic and inorganic monomers or polymers. For example, in certain embodiments, the agents of the invention include biomolecules such as lipids, nucleotides, nucleosides, amino acids, and sugars. In some embodiments, these biomolecules include biopolymers, such as peptides, proteins, polypeptides, enzymes, certain hormones, immunoglobulins, polysaccharides, oligosaccharides, polynucleotides, or oligonucleotides.

The reagents of the invention can be used in many different processes. Representative uses of, for example, nucleotide and nucleoside reagents include, but are not limited to, labeling of oligonucleotides formed by enzymatic synthesis, such as nucleoside triphosphates used in the context of PCR amplification, nicking translation reactions, and Sanger-type oligonucleotide sequencing. For example, in certain embodiments, these reagents are labeled Nucleosides (NTPs) that label a substantially non-fluorescent donor moiety of the invention, such as cytidine, adenosine, guanosine, and thymidine. These reagents are useful in a variety of methods involving oligonucleotide synthesis. In some embodiments these reagents are also labeled nucleotides, such as nucleoside mono-, di-, and triphosphates. More specifically, these reagents include, for example, deoxynucleoside triphosphates (dntps) labeled with donor moieties described herein, such as deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate and deoxyguanosine triphosphate. These reagents can be used as polymerase substrates, for example, in the preparation of dye-labeled oligonucleotides. These reagents also include labeled dideoxynucleoside triphosphates (ddNTPs) (e.g., dideoxynucleoside triphosphate, and dideoxynucleoside triphosphate) or other stop nucleotides that label a substantially non-fluorescent donor moiety of the invention. These reagents can be used, for example, in dye termination sequencing. Examples of uses of oligonucleotide reagents include, but are not limited to, use as nucleic acid sequencing primers, PCR primers, hybridization probes, hairpin probes, and 5' -nuclease probes. Representative uses of polypeptides labeled with substantially non-fluorescent donor moieties described herein include protein structure and conformation studies, receptor/ligand binding assays, and immunoassays. Examples of uses of lipid agents labeled with the donor moieties described herein include lipid distribution and transport assays, membrane fusion assays, and membrane potential sensing assays. In addition, exemplary uses of carbohydrate reagents labeled with substantially non-fluorescent donor moieties described herein include receptor/ligand binding assays and structural assays. Methods of using the agents of the invention are described further below.

In certain embodiments, reagents of the invention comprise, in addition to one or more substantially non-fluorescent donor moieties, one or more acceptor moieties and/or quencher moieties. In other embodiments, a given reagent lacks an acceptor moiety and/or a quencher moiety. By way of further example, FIGS. 1A-F show representative embodiments of the agents of the present invention. More specifically, in each of FIGS. 1A-F, biopolymer 100 is schematically represented by monomeric unit 102. As shown in the embodiment shown in fig. 1A, biopolymer 100 includes a substantially non-fluorescent donor moiety (D)104 attached to a monomeric unit 102. In fig. 1B, the acceptor moiety (a)106 is connected to the monomeric unit 102 of the biopolymer 100 through the substantially non-fluorescent donor moiety (D)104, while in fig. 1C, the substantially non-fluorescent donor moiety (D)104 is connected to the monomeric unit 102 of the biopolymer 100 through the acceptor moiety (a) 106. FIG. 1D shows another way in which substantially non-fluorescent donor moieties (D)104 and acceptor moieties (A)106 are attached to biopolymer 100. FIG. 1E shows that substantially non-fluorescent donor moiety (D)104 and acceptor moiety (A)106 are each attached to the same monomer unit 102. In addition, fig. 1F shows that substantially non-fluorescent donor moieties (D)104, acceptor moieties (a)106, and quencher moieties (Q)108 are each attached to a different monomeric unit 102 of the biopolymer 100. It will be appreciated that the donor moiety, acceptor moiety and quencher moiety are optionally covalently or non-covalently linked to the reagent and/or to each other. For example, in certain embodiments, the moieties are connected to each other and/or to other components of the agent by linker moieties, while in other embodiments, they are not connected to each other or to other components of the agent by linker moieties. It will also be appreciated that the donor moiety, acceptor moiety and quencher moiety may be attached to the polymer via the backbone or any other structural component of the polymer molecule.

Acceptor moiety and quencher moiety

The acceptor moiety of the reagents of the invention is typically capable of absorbing the excitation energy transferred from the substantially non-fluorescent donor moiety and in response fluoresce. In some embodiments, the peak visible absorptions of the substantially non-fluorescent donor moiety and the acceptor moiety differ by about 100nm or greater. Representative major classes of acceptor moieties that can be used include, but are not limited to, xanthene dyes, cyanine dyes, phthalocyanine dyes, and squarylium dyes. Examples of acceptor moieties for dyes belonging to these classes are also described, for example, in U.S. Pat. No. 5,800,996 to Lee et al, titled "ENERGYTRANSFER DYES WITH ENHANCED FLUORESCENCE", granted 9/1 of 1998.

Further by way of example, more specific examples of acceptor moieties that may be used in certain embodiments of the agents of the invention include, but are not limited to, isomers of carboxyfluorescein (e.g., 5 and 6 carboxy groups), 4, 7-dichlorofluorescein, 4, 7-DICHLORORHODAMINE (U.S. Pat. No. 5,847,162 to Lee et al, titled "4, 7-DICHLORORORORORORHODAMINE DYES (4, 7-DICHLORORHODAMINE DYES)", granted 12.8.1998), fluorescein, asymmetric benzoxanthene DYES, isomers of carboxy-HEX (e.g., 5 and 6 carboxy groups), NAN, CI-FLAN, TET, JOE, VIC, ZOE, rhodamine, isomers of carboxyrhodamine (e.g., 5 and 6 carboxy groups), isomers of carboxy R110 (e.g., 5 and 6 carboxy groups), isomers of carboxy-R6G (e.g., 5 and 6 carboxy groups), isomers of N, N ', N' -tetramethylcarboxyrhodamine (e.g., 5 and 6 carboxy groups), MR, Isomers of carboxy-X-Rhodamine (ROX) (e.g., 5 and 6 carboxy), LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, JA-270, CY3, CY3.5, CY5, CY5.5, BODIPYDyes (e.g. FL, 530/550, TR, and TMR), ALEXA FLUORDyes (e.g., 488, 532, 546, 568, 594, 555, 653, 647, 660, and 680), other energy transfer dyes (e.g., BIGDYE)^TMv1 dye, BIGDYE^TMv2 dye and BIGDYE^TMv3 dye), fluorescent dyes (e.g. lucifer yellow), CASCADE BLUEAnd Oregon Green. Further examples of suitable acceptor portionsAs provided for example in the Haugland,Molecular Probes Handbook of Fluorescent Probes and Research Productsversion 9, (2003) and its newer versions. Acceptor moieties are generally readily available from a number of commercial suppliers, including, for example, Molecular Probes, inc. (Eugene, OR, USA), Amersham Biosciences corp. (Piscataway, NJ, USA), and applied biosystems (Foster City, CA, USA).

In certain embodiments, the substantially non-fluorescent donor moiety and the acceptor moiety are not connected to each other by a linker moiety. In other embodiments, the portions are connected to each other by a joint portion. For example, in some of these embodiments, the linker moiety lacks the following structure:

wherein R is₄To a C which is attached to a substantially non-fluorescent donor moiety_1-5Alkyl radical, R₅Selected from NH, S and O, R₆Selected from the group consisting of alkenes, dienes, alkynes, and five or six membered rings having at least one unsaturated bond or a fused ring structure attached to a carbonyl carbon; r₇Comprising a functional group that connects the linker moiety to the acceptor moiety. The linker moiety is described further below.

In some embodiments, the reagents of the invention further comprise one or more quencher moieties. The problem is usually determined by its excitation and emission spectra and the fluorochromes that are paired with it, given whether the fluorochrome is a reporter or a quencher. Fluorescent molecules commonly used as quencher moieties include, but are not limited to, e.g., fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL, and EDANS. Many of these compounds are available from the commercial suppliers mentioned above. Exemplary non-fluorescent Quenchers or dark Quenchers capable of emitting energy absorbed from fluorescent dyes include Black Hole Quenchers commercially available from Biosearch Technologies, Inc. (Novato, Calif., USA)^TMOr BHQ^TM。

In certain embodiments, the reagents of the invention may also include other labeling moieties. Examples of other label moieties include, but are not limited to, e.g., biotin, a weak fluorescent label (Yin et al, (2003)Appl Environ Microbiol.69(7): 3938, Babendrere et al (2003)Anal. Biochem.317(1): 1 and Jankowiak et al, (2003)Chem Res Toxicol.16(3): 304) radioactive, non-fluorescent, colorimetric, chemiluminescent (Wilson et al, (2003)Analyst.128(5): 480 and Roda et al (2003)Luminescence18(2): 72) raman, electrochemical, bioluminescent markers (Kitayama et al, (2003)Photochem Photobiol.77(3): 333, Arakawa et al (2003)Anal.Biochem.314(2): 206 and Maeda (2003)J.Pharm.Biomed.Anal.30(6): 1725) and alpha-methyl-PEG labeling reagents such as those described in the following patent applications: bodepudi et al, U.S. provisional patent application No. 60/428,484, titled "DETECTABLE Label tape dispensing crystals and METHODS OF USE THEREOF", filed 11, 22/2002.

Joint part

There are a wide variety of linker moieties available for linking other moieties (e.g., substantially non-fluorescent donor moieties, acceptor moieties, and quencher moieties) to the biomolecules and other reagents of the invention, which are well known to those skilled in the art. Linker moieties are generally structurally and electronically suitable for attachment to a given biomolecule in a spatial arrangement. For example, the linker moiety optionally includes, for example, an ether, thioether, carboxamide, sulfonamide, urea, urethane, hydrazine, or other moiety. By way of further example, the linker moiety typically comprises from about 1 to about 25 non-hydrogen atoms selected from, for example, C, N, O, P, Si and S, including, for example, ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide linkages, and virtually any combination of aromatic or heteroaromatic linkages. For example, in some embodiments, the linker moiety comprises a combination of a single carbon-carbon bond and a carboxamide or thioether bond. The linear segment of the linker typically contains from about 3 to about 15 non-hydrogen atoms, although longer linear segments are optionally employed. Certain of these exemplary types of joints are described further below.

By way of further example, non-limiting examples of linker moieties include substituted (e.g., functionalized) or unsubstituted groups. More specifically, exemplary linkers include imidazole/biotin linkers, polymethylene groups, arylene groups, alkylarylene groups, arylenealkyl groups, arylthio groups, amidoalkyl groups, alkynylalkyl groups, alkenylalkyl groups, alkyl groups, alkoxy groups, thio groups, aminoalkyl groups, morpholine-derivatized phosphoric acids, peptide nucleic acids (e.g., N- (2-aminoethyl) glycine), and disulfide groups. In some embodiments, when the complementary functionality or attachment site to the biomolecule is an amine, the linker moiety is an isothiocyanate, sulfonyl chloride, 4, 6-dichlorotriazinylamine, succinimidyl ester or another reactive formate. Optionally, when the complementary functionality is a thiol group, the linker moiety is a maleimide, haloacetyl or iodoacetamide. In certain embodiments, the linker moiety is an activated N-hydroxysuccinimide (NHS) ester formed from a carboxyl group on the donor moiety, acceptor moiety, or quencher moiety that is capable of reacting with an aminohexyl group of a particular biomolecule. Some of these and other linkers are further described in, for example, the following patents: haugland et al, us patent 6,339,392, us patent 5,047,519, Hobbs, jr. et al, us patent 4,711,958, Iizuka et al, us patent 5,175,269, Stavrianopoulos et al, us patent 4,711,955, Ward et al, us patent 5,241,060, Engelhardt et al, us patent 5,328,824, and us patent publication 2002/0151711, Khan et al. Additional details regarding biomolecular markers and linker moieties are provided in, for example, Hermanson,Bioconiugate Techniqueselsevier Science (1996) and Haugland,Molecular Probes Handbook of Fluorescent Probes and Research Productsversion 9, (2003) and its newer versions.

In certain embodiments, suitable linkers comprise photocleavable moieties such as 2-nitrobenzyl moieties, α -substituted 2-nitrobenzyl moieties (e.g., 1- (2-nitrophenyl) ethyl moieties), 3, 5-dimethoxybenzyl moieties, thiohydroxamic acids, 7-nitroindoline moieties, 9-phenylxanthyl moieties, benzoin moieties, hydroxybenzoylmethyl moieties, and NHS-ASA moieties. Photo-cleavable linkers are further described, for example, in U.S. patent publication No. 2003/0099972 to Olejnik et al. In some embodiments, the linker comprises a metal atom, such as a platinum atom. Such linkers are further described, for example, in U.S. Pat. No. 5,714,327 to Houthoff et al. There are a variety of different lengths of linker commercially available from a variety of suppliers, including, for example, Operon Biotechnologies, Inc. (Huntsville, AL, USA), BD Biosciences Clontech (Palo Alto, Calif., USA), and Molecular Biosciences (Boulder, CO, USA).

In the case of nucleosides, nucleotides, and nucleic acids, the substantially non-fluorescent donor moiety, acceptor moiety, and quencher moiety are optionally linked, e.g., via an amide, ester, thioester, ether, thioether, carbon-carbon, or other type of covalent bond, to, e.g., a carbocyclic, heterocyclic, or aryl group of a nucleoside or nucleotide (e.g., via the C of a pyrimidine⁵Cytidine N⁴N of purine⁷N of adenosine⁶C of purine⁸Or other attachment sites known in the art). Additionally or alternatively, the specified moiety is linked to the sugar moiety of the nucleoside or nucleotide (e.g., ribose) or an analog thereof (e.g., carbocyclic ring) and/or the phosphate group of the nucleotide, for example, by a covalent bond such as an amide, ester, thioester, ether, thioether, carbon-carbon or other bond. Covalent bonds are typically formed in reactions between these moieties and electrophilic and nucleophilic groups of the nucleoside or nucleotide. In certain embodiments, the substantially non-fluorescent donor moiety, acceptor moiety, and quencher moiety are directly conjugated to the nucleotide (e.g., via a single, double, triple, or aromatic carbon-carbon bond, or via a carbon-nitrogen bond, a nitrogen-nitrogen bond, a carbon-oxygen bond, a carbon-sulfur bond, a phosphorus-oxygen bond, and a phosphorus-nitrogen bond).

In some embodiments, the linker moiety is an alkynylamido or enamido linkage, and the linkage between the substantially non-fluorescent donor moiety, acceptor moiety or quencher moiety and the nucleotide base is formed by reacting an activated NHS ester of a particular said moiety with an alkynylamino-, alkynylethoxyamino-or alkenylamino-derivatized base of the nucleotide. The resulting linkage may include, for example, propargyl-1-ethoxyamido (3- (amino) ethoxy-1-propynyl), 3- (carboxy) amino-1-propynyl, or 3-amino-1-propyn-1-yl.

By way of further example, the synthesis of alkynylamino-derivatized nucleosides is also described, for example, in Hobbs (1989) "Palladium-catalyzed synthesis of alkylaminonucleosides. A univalent linker for nucleic acids",J.Org.Chem.54: 3420-3422. Briefly, alkynylamino-derivatized nucleosides are formed by: the appropriate halogenated dideoxynucleosides (typically 5-iodo-pyrimidine and 7-iodo-7-deazapurine dideoxynucleosides) and Cu (I) were placed in a flask, purged with argon to remove air, dry DMF was added, followed by the addition of alkynylamine, triethylamine and Pd (0). The reaction mixture may be stirred for several hours or until thin layer chromatography indicates that the halogenated dideoxynucleoside is consumed. When unprotected alkynylamines are used, the alkynylaminoiucleosides can be isolated by: the reaction mixture was concentrated and subjected to silica gel chromatography using an eluting solvent containing ammonium hydroxide to neutralize the hydrohalide produced in the coupling reaction. When the protected alkynylamine is used, methanol/methylene chloride may be added to the reaction mixture, and then a strongly basic anion exchange resin in the bicarbonate form is added. The resulting slurry was then stirred for about 45 minutes, filtered, and the resin washed with additional methanol/dichloromethane. The combined filtrates may be concentrated and purified by flash silica gel chromatography with a methanol-dichloromethane gradient. The triphosphate can be obtained using standard techniques well known to those skilled in the art.

Biomolecule preparation

The biomolecules or other reagents of the invention may be prepared by any suitable method, including synthetically produced or obtained from natural sources (e.g., prior to labeling). For example, a substantially non-fluorescent donor moiety, acceptor moietyThe synthesis of oligonucleotides, and/or quencher moieties, can be accomplished using any of a variety of known oligonucleotide labeling techniques. For example, the labeled oligonucleotide can be synthesized enzymatically, e.g., with a DNA polymerase or ligase, or chemically, e.g., with the phosphoramidite method or the phosphite triester method (Herdewijn,Oligonucleotide Synthesis：Methods and Applicationshumana Press (2005), Gait (eds),Oligonucleotide Synthesisoxford University Press (1984), Vorbruggen et al,Handbook of Nucleoside Synthesis，John Wiley &sons, Inc. (2001) and Hermanson,Bioconjugate Techniqueselsevierscience (1996)). The label can be introduced during enzymatic synthesis, for example, with labeled nucleoside triphosphate monomers, or during chemical synthesis with labeled non-nucleotides or nucleotide phosphoramidites, or can be introduced after synthesis. Methods for synthesizing phosphoramidites comprising substantially non-fluorescent donor moieties are also illustrated in the examples provided below.

Exemplary methods for enzymatically synthesizing labeled oligonucleotides include denaturing the template or target nucleic acid and annealing primers to the template. Typically, a mixture of deoxynucleoside triphosphates (e.g., dGTP, dATP, dCTP, and dTTP) is added to a reaction mixture in which at least a portion of one of the deoxynucleotides is labeled with a substantially non-fluorescent donor moiety, acceptor moiety, and/or quencher moiety as described herein. Next, a nucleotide incorporating catalyst such as a polymerase is typically added to the reaction mixture under conditions in which the enzyme is active. The labeled oligonucleotide is formed by incorporation of labeled deoxynucleotides during polymerase chain synthesis. In an alternative enzymatic synthesis method, two primers are used instead of one, where one primer is complementary to a portion of one strand of the target double-stranded nucleic acid and the other primer is complementary to a portion of the other strand of the nucleic acid. The polymerase employed in the present method is typically a thermostable enzyme, and the reaction temperature is typically cycled between a denaturation temperature and an extension temperature to achieve synthesis of a labeled complementary strand of the target nucleic acid by PCR (Edwards et al (editors),Real-Time PCR：An Essential Guidehorizon Scientific Press (2004), Innis et al (eds.),PCR Strategies，ElsevierScience &technology Books (1995) and Innis et al (eds.),PCR Protocols，Academic Press(1990)。

labeled oligonucleotides prepared by chemical synthesis are typically generated by the phosphoramidite method, although other methods may optionally be used. Phosphoramidite-based synthesis is often performed with the growing oligonucleotide chain attached to a solid support so that excess reagents in the liquid phase can be easily filtered off. This eliminates the need for additional purification steps between cycles.

An exemplary solid phase oligonucleotide synthesis cycle using the phosphoramidite method is briefly described below: the solid support comprising the protected nucleotide monomer is typically first treated with an acid (e.g., trichloroacetic acid) to remove the 5' -hydroxy protecting group and free the hydroxy group for subsequent coupling reactions. The activated intermediate is then typically formed by simultaneously adding the protected phosphoramidite nucleoside monomer and a weak acid (e.g., tetrazole) to the reaction. The weak acid protonates the nitrogen of the phosphoramidite to form a reactive intermediate. The addition of nucleosides to the growing nucleic acid strand is typically completed within 30 seconds. Thereafter, a capping step is typically performed to terminate any oligonucleotide chains where no nucleotide incorporation has occurred. The capping can be carried out, for example, with acetic anhydride and 1-methylimidazole. The internucleotide linkages are then converted from phosphites to more stable phosphotriesters by oxidation with, for example, iodine as the oxidant and water as the oxygen donor. After oxidation, the cycle is repeated by removing the hydroxyl protecting group with a protic acid (e.g., trichloroacetic acid or dichloroacetic acid) until chain extension is complete. After synthesis, the synthesized oligonucleotide is cleaved from the solid support, usually with a base such as ammonium hydroxide or tert-butylamine. The cleavage reaction also removes any phosphate protecting groups (e.g., cyanoethyl). Finally, the protecting groups on the exocyclic amine of the base and the hydroxyl protecting group on the labeling moiety are removed by treating the oligonucleotide solution at elevated temperature (e.g., up to about 55 ℃) under basic conditions.

A description of the chemical principles used to form oligonucleotides by the PHOSPHORAMIDITE method is also provided, for example, in U.S. Pat. No. 4,458,066 to Caruthers et al, entitled "PROCESS FOR REPARING POLYNUCLEOTIEDES," granted on 3.7.1984, AND U.S. Pat. No. 4,415,732 to Caruthers et al, entitled "PHOHORAMIDE COMPOSITIONS AND PROCESSES (PHOSPHORAMIDITE Compounds AND methods)," granted on 15.11.1983.

Any phosphoramidite nucleoside monomer can be labeled with a substantially non-fluorescent donor moiety, acceptor moiety, and/or quencher moiety as described herein. In certain embodiments, if the 5' -end of the oligonucleotide is to be labeled, a labeled non-nucleotidic phosphoramidite may be used during the final condensation step. If the internal position of the oligonucleotide is to be labeled, a labeled nucleotide phosphoramidite can be used during any of the condensation steps. In addition, oligonucleotides can also be labeled at almost multiple positions after synthesis (Eckstein et al (eds.),Oligonucleotides and Analogues：A Practical Approachoxford University Press (1992), Chu et al, (1983) "Derivatification of unprotected polynucleotides",Nucleic Acids Res.11(18): 6513-6529 and Smith et al, U.S. Pat. No. 5,118,800, entitled "Oligonucleotides having primary amino group in the terminal nucleotide", 1992, 6.2.months. Further by way of example, the Oligonucleotide may also be labeled on its phosphodiester backbone (Eckstein et al, (1992), supra) or labeled at the 3' -end (Nelson et al, (1992) "Oligonucleotide labeling methods.3.direct labeling of oligonucleotides a novel, non-nucleosidic, 2-aminobutyl-1, 3-propanediol backbone" (Oligonucleotide labeling method. 3.direct labeling of oligonucleotides using a novel non-nucleoside 2-aminobutyl-1, 3-propanediol backbone) ",Nucleic Acids Res.20(23): 6253-functional controlled pore glass (MF-CPG) reagent-labeled synthesized oligonucleotide 3 'end method in solid phase oligonucleotide synthesis, granted on 28. 3.1995 and Nelson's U.S. Pat. No. 5,141,813 entitled "Multifunctional controlled pore glass reagent for solid phase oligonucleotide synthesis", granted on 25. 8.1992.

In certain embodiments, modified nucleotides are included in labeled oligonucleotides (e.g., probes or primers) described herein. For example, the introduction of a modified nucleotide position into an oligonucleotide sequence can, for example, increase the melting temperature of the oligonucleotide. In some embodiments, this results in greater sensitivity than a corresponding unmodified oligonucleotide, even in the presence of one or more mismatches in sequence between the target nucleic acid and the particular oligonucleotide. Exemplary modified nucleotides that may be substituted or added to the oligonucleotide include, for example, C5-ethyl-dC, C5-methyl-dC, C5-ethyl-dU, 2, 6-diaminopurine, C5-propynyl-dC, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2 '-O-methyl Ribo-U, 2' -O-methyl Ribo-C, 8-aza-dA, 8-aza-dG, 7-deaza-dA, 7-deaza-dG, N4-ethyl-dC and N6-methyl-dA. Further by way of example, other examples of modified oligonucleotides include oligonucleotides with one or more LNAs^TMA monomeric oligonucleotide. Nucleotide analogs such as these are also described, for example, in the following patents: U.S. Pat. No. 6,639,059 to Kochkine et al, titled "SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES([2.2.1]Synthesis of bicyclic nucleosides) ", granted on 28.10.2003, Skoov U.S. Pat. No. 6,303,315 titled" ONE STEP SAMPLE PREPARATIONS AND DETECTION OF OFNUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES (ONE-step sample PREPARATION AND DETECTION of nucleic ACIDS IN COMPLEX BIOLOGICAL SAMPLES)Test) ", issued on 16/10/2001 and issued by Kochkine et al, U.S. patent application publication No. 2003/0092905 entitled" SYNTHESISOF [2.2.1]BICYCLO NUCLEOSIDES([2.2.1]Synthesis of bicyclic nucleosides) ", published 5/15/2003. Including LNA^TMMonomeric oligonucleotides can be obtained, for example, from Exiqon A/SIs commercially available. Additional oligonucleotide modifications are mentioned herein, including in the definitions above.

By way of further example, virtually any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) can be custom-made or standard-made from any of a number of commercial sources, such as The Midland Certified Reagent Company, inc. (Midland, TX, USA), Operon Biotechnologies, inc. (Huntsville, AL, USA), proligo llc (Boulder, CO, USA), and many others.

Reagents of the invention may also include proteins and peptides labeled with substantially non-fluorescent donor moieties, acceptor moieties, and/or quencher moieties as described herein. The peptide is optionally synthesized and/or labeled using any known technique. For example, methods of preparing peptides with C-terminal alcohols typically involve solution phase synthesis or solid phase peptide synthesis. Solid phase methods generally employ covalent attachment of the starting material to a solid support (e.g., polystyrene or polyethylene glycol) via a suitable linker moiety well known to those skilled in the art. The C-terminal anchoring strategy for peptide synthesis based on benzyl and benzhydrylamine linkers used in Boc/Bzl (tert-butoxycarbonyl/benzyl) chemistry and the more labile alkoxybenzyl or 2, 4-dimethoxybenzhydrylamine linkers used in the Fmoc/tBu (fluorenylmethoxycarbonyl/tert-butyl) protocol was optionally applied to the synthesis of peptides with free carboxyl or carboxyamide termini. Various aspects of peptide synthesis are also described, for example, in Merrifield (1963)J.Am.Chem.Soc.85: 2149, Larsen et al, (1993)J.Am.Chem.Soc.115: 6247 Smith et al (1994)J. Peptide Protein Res.44: 183, O' Donnell et al, (1996)J.Am.Chem.Soc.118：6070，Mitchell et.al.(1976)J.Am.Chem.Soc.98：7357-7362，Matsueda et.al.(1981)Peptides 2：45-50，Wang(1972)J.Am.Chem.Soc.95：1328-1333，Rink(1987)Tet.Let.28: 3787-3790, Lloyd-Williams et al,Chemical Approaches to the Synthesis of Peptides and Proteins，CRCPress(1997)，Jones，Amino Acid and Peptide Synthesis2 nd edition, Oxford university Press (2002) and Howl,Peptide Synthesis and Applicationshumana Press (2005). Additionally, custom peptides and proteins may also be ordered from various commercial suppliers, including, for example, Sigma-Genesis Co. (The Woodlands, TX, USA), Biopeptide Co., LLC (San Diego, Calif., USA), and Invitrogen Corp. (Carlsbad, Calif., USA).

In certain embodiments, the reagents of the invention are immunoglobulins or antibodies that include a substantially non-fluorescent donor moiety, acceptor moiety and/or quencher moiety as described herein. Antibodies suitable for use in these embodiments of the invention may be prepared and labeled by conventional methods and/or genetic engineering. Antibody fragments may be derived, for example, from light and/or heavy chains (V)_HAnd V_L) Or simultaneously from V (including hypervariable regions)_HRegion and V_LAnd (3) genetic engineering of the region. For example, the term "immunoglobulin" or "antibody" as used herein includes polyclonal and monoclonal antibodies and biologically active fragments thereof, including "monovalent" antibodies (Glennie et al, (1982)Nature295: 712) (ii) a Fab proteins, including Fab 'and F (ab') which aggregate covalently or non-covalently₂A fragment; individual light or heavy chains, usually heavy and light chain variable regions (V)_HRegion and V_LRegion), more typically including a hypervariable region (also known as V)_HRegion and V_LComplementarity Determining Regions (CDRs) of the regions); f_cA protein; "hybrid" antibodies capable of binding more than one antigen; a constant region-variable region chimera; "complex" immunoglobulins with heavy and light chains of different origins; "altered" antibodies with improved specificity and other properties are prepared by standard recombinant techniques, mutagenesis techniques or other directed evolution techniques well known in the art. MarkingThe immunoglobulins of (a) can be used in a variety of well-known assays, including, for example, fluorescent immunoassays.

Other exemplary reagents of the invention include lipids and carbohydrates comprising substantially non-fluorescent donor moieties, acceptor moieties and/or quencher moieties as described herein. Various aspects of lipid and/or carbohydrate chemistry and synthesis are also described, for example, in Tyman (Ed.),Surfactants in Lipid Chemistry：Recent Synthetic，Physical，and Biodegradative Studiesthe Royal Society of Chemistry (1992), Gurr et al,Lipid Biochemistry5 th edition, Iowa State Press (2001), Min Kuo et al (eds.),Lipid Biotechnologymarcel Dekker (2002), Ogura et al (eds.),Carbohydrates-Synthetic Methods and Applications in Medicinal ChemistryVCH Publishers, Inc. (1993), Derek Horton et al (eds.),Trends in Synthetic Carbohydrate Chemistryamerican Chemical Society (1989), Gunstone et al,Lipid Handbook2 nd edition, CRC Press (1994), Scherz et al,Analytical Chemistry of Carbohydrates，John Wiley &sons, inc. (2002), Boons (eds),Carbohydrate Chemistry，Chapman &hall (1997), Davis et al,Carbohydrate Chemistry，Oxford University Press(2002)。

v. reaction mixture

The present invention also provides a number of different reaction mixtures that can be used in a variety of applications. For example, in some embodiments, the reaction mixture is used to perform a homologous (homologous) amplification/detection assay (e.g., real-time PCR monitoring), a nucleic acid sequencing procedure, a biopolymer synthesis protocol (e.g., peptide synthesis or oligonucleotide synthesis), or a biopolymer labeling reaction. Many of these applications are described further below or otherwise mentioned herein.

In certain embodiments, the reaction mixtures of the present invention include selected amounts of nucleotides, primers, and/or probes. Typically, one or more of these nucleotides, primers and/or probes is labeled with at least one substantially non-fluorescent donor moiety, acceptor moiety and/or quencher moiety. The nucleotides of these reaction mixtures are typically extendable nucleotides and/or terminating nucleotides, for example, for use in nucleic acid amplification reactions or nucleic acid sequencing reactions. Almost any terminator nucleotide known to those skilled in the art may optionally be used. Some exemplary TERMINATOR NUCLEOTIDEs useful in some embodiments are also described, for example, in U.S. patent 5,273,638 to Konrad et al, entitled "NUCLEOTIDE sequencing with matched concentrations OF DIDEOXYNUCLEOTIDE TERMINATORs," granted on 28.12.1993, U.S. patent application No. 10/879,493 to Gelfand et al, entitled "2 '-TERMINATOR NUCLEOTIDEs-relatedends AND SYSTEMS (2' -TERMINATOR related methods and systems)," filed on 28.6.2004, and U.S. patent application No. 10/879,494 to bodeputidi et al, entitled "SYSTHESIS AND NUCLEOTIDEs OF 2 '-TERMINATOR NUCLEOTIDEs (synthesis and composition OF 2' -TERMINATOR NUCLEOTIDEs)", filed on 28.6.2004, 28.6.28.2004. The probes included in these reaction mixtures typically include, for example, hybridization probes, 5' -nuclease probes, and/or hairpin probes.

In addition, the reaction mixture also typically includes a variety of reagents that can be used to perform a nucleic acid amplification or detection reaction (e.g., real-time PCR monitoring or 5' -nuclease assay) or a nucleic acid sequencing reaction. Exemplary types of these other reagents include, for example, template or target nucleic acids, biocatalysts that catalyze nucleotide incorporation (e.g., DNA polymerases), buffers, salts, amplicons, glycerol, metal ions, Dimethylsulfoxide (DMSO), and poly rA. Nucleic acid amplification and detection and other methods are also described further below.

The reaction mixture is typically generated by combining the selected nucleotides, primers and/or probes described above with an amount of other reagents sufficient to carry out the particular application selected. The amount of reagents to be included in a given reaction mixture will be apparent to those skilled in the art from the selected method to be performed. However, for example, primer nucleic acids and extendable nucleotides (e.g., four dNTPs (dGTP, dCTP, dATP, dTTP)) are each typically present in these reaction mixtures in large molar excess. Probes and primers useful in the reaction mixtures of the present invention are described herein. Suitable extendable and/or terminator nucleotides are readily available from many different commercial suppliers, including, for example, Roche Diagnostics Corporation (Indianapolis, IN, USA), Amersham Biosciences Corp. (Piscataway, NJ, USA), and Applied Biosystems (Foster City, CA, USA).

Biocatalysts that catalyze nucleotide incorporation for use in the reaction mixtures and other aspects of the invention include enzymes such as polymerases, terminal transferases, reverse transcriptases, telomerase, and polynucleotide phosphorylases. For example, in certain embodiments, the enzyme comprises 5 '-3' nuclease activity, 3 '-5' exonuclease activity, and/or is a thermostable enzyme. The enzyme is optionally derived from a microorganism such as: thermus antarikinii, Thermus aquaticus (Thermus aquaticus), Thermus caldophilus, Thermus thermophilus (Thermus chalarphilus), Thermus flavus (Thermus longipedicularis), Thermus lactis, Thermus aeromonas (Thermus osiformimai), Thermus ruber (Thermus ruber), Thermus rubens, Thermus aquaticus (Thermus scodottus), Thermus sylvestris (Thermus Silverana), Thermus specz 05, Thermus specs 17, Thermus thermophilus (Thermus thermophilus), Thermus marinus (Thermogenesses), Thermus thermophilus (Thermus mariticus), Thermus thermophilus (Thermus thermophilus), Thermus thermophilus (Thermoascus), Thermoascus thermoascus sp (Thermoascus aeromonas), Thermoascus thermoanaerobacter (Thermoascus aerobacter), Thermoascus anaerobacter sp, and Thermoascus lipomyces.

In certain embodiments, additional reagents may also be added to the reaction mixtures of the present invention. For example, the reaction mixture also optionally includes, e.g., a pyrophosphatase (e.g., a thermostable pyrophosphatase) for minimizing pyrophosphorolysis, e.g., uracil N-glycosylase (UNG) (e.g., thermostable UNG) to prevent carryover contamination.

Additional exemplary reaction mixtures of the invention include biopolymer synthesis reagents (e.g., nucleic acid synthesis reagents such as phosphoramidites or polypeptide synthesis reagents) described herein. These reaction mixtures are typically used in biopolymer synthesis processes mentioned herein or otherwise well known to those skilled in the art.

A number of reaction mixtures of biomolecules and other reagents suitable for use in the present invention are also described, for example, in Ausubel et al (eds.)Current Protocols in Molecular BiologyVolume I, II and III, (1997) (Ausubel 1), Ausubel et al (eds.),Short Protocols in Molecular Biology：A Compendium of Methods from Current Protocols in Molecular Biology5 th edition, John Wiley&Sons, inc. (2002) (Ausubel 2), Sambrook et al,Molecular Cloning：A Laboratory Manual3 rd edition, Cold spring Harbor Laboratory Press (2000) (Sambrook), Berger and Kimmel,Guide to Molecular Cloning Techniques：Methods in Enzymologyvorbruggen et al, Academic Press, Inc. (Berger),Handbook of Nucleoside Synthesis，Organic Reactions Series，#60，John Wiley &sons, inc. (2001), Gait (eds)Oligonucleotide SynthesisOxford university Press (1984), Hames and Higgins,Nucleic Acid Hybridizationpractical Applirocach Series, Oxford University Press (1997) and Hames and Higgins (eds)Transcription and Translation，Practical Approach Series，OxfordUniversity Press(1984)。

Methods of using biomolecules comprising substantially non-fluorescent donor moieties

The invention also provides methods of using the labeled biomolecules described herein. For example, in some embodiments, these biomolecules are used to perform assays involving the detection of target nucleic acids or other biomolecules, e.g., in order to provide diagnostic information, genetic information, or other information about the subject from which the targets are derived. The use of substantially non-fluorescent donor moieties in these methods typically results in a reduction in background fluorescence compared to procedures employing conventional donor moieties. Thus, this will generally improve the performance characteristics, such as sensitivity and dynamic range, of the particular assay in which these substantially non-fluorescent donor moieties are used. These aspects are also illustrated in the examples provided below.

The biomolecules described herein are optionally used or suitable for almost any application involving the transfer of energy to effect analyte detection. Examples of types of applications related to nucleic acids include analysis of nucleic acid structure and conformation, real-time PCR assays and SNP detection (Myakishev et al, (2001) 'High-throughput SNP genotyping by allele-specific PCR with universal energy transfer marker primers)',Genome Res11: 163-169, Lee et al, (1999) "Seven-color homologous detection of sixPCR products," Seven-color, homogenetic detection of six PCR products,Biotechniques27: 342-349, Thelwell et al, (2000) "Mode of action and application of Scorpion primers to mutation detection",Nucleic Acids Res28: 3752-3761, Whitcombe et al, (1999) "Detection of PCR products using self-binding assays and fluorescence)",Nat Biotechnol17: 804-,Genome Res6: 986. sup. 994, Nazarenko et al, (1997) "A closed tube format for amplification and detection of DNA based on energytransfer",Nucleic Acids Res25: 2516 and 2521); detection of nucleic acid hybridization (Parkhurst et al, (1995) "kinetic by fluorescence resonance energy transfer-labeled oligonucleotide: hybridization to the oligonucleotide and to single-stranded DNA (kinetic-resonance energy transfer using double-labeled oligonucleotides)Study: hybridization with oligonucleotide complementary sequence and with single-stranded DNA) ",Biochemistry34: 285 ℃ 292, Tyagi et al, (1996) "Molecular beacons: probes which fluoresce upon hybridization),Nat Biotechnol14: 303-308, Tyagi et al, (1998) "multicolumorganic beacons for allele discrimination",Nat Biotechnol16: 49-53, Sixou et al, (1994) "intracellularly oligomeric hybridization detected by Fluorescence Resonance Energy Transfer (FRET) hybridization",Nucleic Acids Res22: 662-668 and Cardullo et al, (1988) "detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer",Proc Natl Acad Sci USA85: 8790-8794); primer extension assay to detect mutations (Chen et al, (1997) "Fluorescence energy transfer detection as an homologous DNA diagnostic method)",Proc Natl Acad Sci USA94: 10756 — 10761); and automatic DNA sequencing (Woolley et al (1995) 'Ultra-high-speed DNA sequencing using capillary electrophoresis chips)',Anal Chem67: 3676 3680, Hung et al, (1998) "fluorescence of fluorescence energy transfer primers with differential Donor-acceptor dye combinations",Anal Biochem255: 32-38 and Ju et al (1995) "Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis",Proc Natl Acad Sci USA92：4347-4351)。

examples of the types of applications associated with proteins include the analysis of protein structure and conformation (Xing et al, (1995) "Internal movement in myostatin subfragment 1 detected by fluorogenic response energy transfer (internal motion in myosin subfragment 1 detected by fluorescence resonance energy transfer) ",Biochemistry34: 6475 6487, Luo et al, (1998) "Localization of Cys133 of rabbit skeletal troponin-I with therapy to troponin-C by resonance energy transfer",Biophys J74: 3111-3119, Erickson et al, (1995) "Use of a response energy transduction polypeptide binding site of the guanine nucleotide transduction polypeptide on the gamma site of the cyclic GMP phosphodiesterase using resonance energy transfer",Biochemistry34: 8693-8700 and Taniguchi et al, (1993) "Reversible changes in the fluorescence energy conversion of the fluorescence energy transduction of interaction intermediates in the reaction-labeled DNA-labeled (Na +, K +) -ATPase (Reversible change in structure accompanied by fluorescence energy transfer of reaction intermediates in the probe-labeled (Na +, K +) -ATPase)",J Biol Chem268: 15588 and 15594); analysis of the spatial distribution and assembly of protein complexes (Moens et al, (1994) "Determination of the radial correlation of Cys-374 in F-action using fluorescence spectroscopy transfer spectroscopy: effect of Cys-374 in F-actin: the effect of phalloidin on polymerase assembly",Biochemistry33: 13102-13108, Watson et al, (1995) "Macromolecular arrangement in the amino-acyl-tRNA. interaction factor Tu. GTP tertiary complex. fluorescence energy transfer study",Biochemistry34: 7904-dimerize in phospholipid bilayers: a stress energy transduction (dimerization of the helical transmembrane domain of glycophorin A in a phospholipid bilayer: resonance energy transfer study) ",Biochemistry33: 5539-5544 and Matyus (1992) "fluorescence resonance energy generation transfer on cell surfaces. A spectroscopic probes for determining protein interactions",J Photochem Photobiol12: 323-337); analysis of receptor/ligand interactions (Berger et al, (1994) "Complex molecular mechanisms for binding of dihydropyridine to L-type Ca (2+) -channels as secreted by fluorogenic responses transfer",Biochemistry33: 11875-^TMbinding to G protein-coupled receptors (measurement of fluoropeptide using polarization detection)^TMBinding to a G protein-coupled receptor) ",J Recept Signal Transduct Res22: 333-343 and Poo et al, (1994) "restriction of CD3 triggerransmembrane restriction between LFA-1 and biological microfluorides in an enzymatic T cell cycle derived from molecular encapsulation cells: quantitative resonance energy transfer microscopy (ligation of CD3 triggers membrane proximity between LFA-1 and cortical microfilaments in cytotoxic T cell clones derived from tumor-infiltrating lymphocytes)',J Cell Physiol159: 176-180); and immunoassays (Morrison (1988) "Time-resolved detection of energy transfer: theory and use in immunoassays)",Anal Biochem174: 101-120 and Khanna et al, (1980) "4 ', 5' -dimethyl-6-carboxfluorescein: a novelddipole-dipole coupled fluorescence transfer access processor used for fluorescence immunoassays (4 ', 5' -dimethoxy-6-carboxyfluorescein: a novel dipole-dipole coupled fluorescence energy transfer acceptor useful for fluorescence immunoassay) ",Anal Biochem108：156-161)。

examples of the types of applications related to lipids and other types of applications include analysis of lipid distribution and transport (Gutierrez-Merino et al (1995) "Preferential distribution of the fluorescent lipid distribution of the fluorescent pigment probes NBD-phospholipid cholesterol and a polar-phospholipid amide in the exo-phospholipid ether of the acetyl-phospholipid receptor-rich membranes from Toedo marmorata (Preferential distribution of the fluorescent lipid probes NBD-phosphatidylcholine and rhodamine-phosphatidylethanolamine in the outer surface leaflets of acetylcholine receptor-rich membranes in skate)",Biochemistry34: 4846-,Biochemistry31: 2865 2873); membrane fusion assays (Pecheur et al, (1998) "Membrane fusion induced by 11-mer ionic and cationic peptides: a Structure-function study of 11-mer anionic and cationic peptide-induced Membrane solutions",Biochemistry37: 2361-,Biochim Biophys Acta1189: 175-180); membrane potential sensing assays (Gonzalez et al, (1995) "Voltage sensing by fluorescence spectroscopy induction cells (Voltage sensing by fluorescence resonance energy transfer in single cells)",Biophys J69: 1272-1280); analysis of Fluorogenic protease substrates (Kurth et al, (1998) "Engineering the S1' subset of trypsin: design of a protease in white bacteria in two bases residues (Engineering of pancreas in the absence of protease in the presence of a protease in the sampleS1' subsite of protease: design of proteases capable of cleaving between dibasic residues) ",Biochemistry37: 11434 and Gulnik et al, (1997) "Design of sensitive fluorescent substrates for human cathepsins D",FEBS Lett413: 379-384); analysis of Cyclic AMP indicators (Adams et al (1991) "fluorescence ratio imaging of cyclic AMP in single cells",Nature349: 694-697) and analysis of Zinc finger indicators (Godwin et al, (1996) "fluorescent Zinc probes Based on Metal-Induced Peptide Folding",J Am Chem Soc118: 6514) (ii) a And analysis for detecting the interaction of single molecules (Ha et al, (1996) "binding the interaction between single molecules: fluorescence resonance energy transfer between single donor and single acceptor and a single receptor (detection of the interaction between two single molecules)",Proc Natl Acad Sci USA93：6264-6268)。

further by way of example, examples of general types of nucleic acid analysis techniques that may be used or adapted for analysis of target nucleic acids in or from, for example, reaction mixtures of the present invention include various nucleic acid amplification assays. Nucleic acid amplification assays have in common that they are generally designed to amplify a nucleic acid sequence that is characteristic of the organism to be detected. Nucleic acid amplification assays generally have greater sensitivity than other nucleic acid analysis methods. This sensitivity is generally attributed to the ability of these assays to produce positive signals from as few as a single copy of the target nucleic acid, and this sensitivity can be further improved with the substantially non-fluorescent donor moieties described herein. Amplification methods that are optionally used or suitable for detecting target nucleic acids include, for example, various polymerase, ligase, or reverse transcriptase-mediated amplification methods, such as Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), and/or reverse transcription PCR (RT-PCR). Further details regarding these and other amplification methods, as well as the various methods of sample preparation for these assays, can be found in any of a variety of standard texts, including, for example, Berger, Sambrook, Ausubel 1 and 2, and Innis, as noted above.

The various commercial nucleic acid amplification assays optionally suitable for use in the reagents and methods of the invention typically differ in their amplification method and their target nucleic acid sequence. Examples of such commercial assays include hybridization probe assays (e.g., using LightCycler)System) and AMPLICOR using Polymerase Chain Reaction (PCR)And COBAS AMPLICORMeasurement (Roche diagnostics Corporation, Indianapolis, IN, USA); LCx using Ligase Chain Reaction (LCR)Assays (Abbott Laboratories, Abbott Park, IL, USA); BDProbeTec using Strand Displacement Amplification (SDA)^TMET test (Becton, Dickinson and Company, Franklin Lakes, n.j., USA); and APTIMA using transcription-mediated amplification (TMA)^TMAssay (Gen-Probe, Inc., San Diego, Calif., USA). Nucleic acid amplification and detection are described further below.

For example, in certain embodiments, the 5 '-nuclease probes of the invention are used in a variety of 5' -nuclease reactions. Many 5' -nuclease assays are known to those of skill in the art. Examples OF such reactions are also described, FOR example, in U.S. Pat. No. 6,214,979 to Gelfand et al, entitled "HOMOGENEEOUS ASSAY SYSTEM (HOMOGENEOUS assay System)", issued on day 10/4/2001, U.S. Pat. No. 5,804,375 to Gelfand et al, entitled "REACTINOXTURES FOR DETECTION OF TARGET NUCLEIC ACIDS (reaction mixture FOR detecting TARGET NUCLEIC ACIDS)", issued on day 8/9/1998, U.S. Pat. No. 5,487,972 to Gelfand et al, entitled "NUCLEIC ACID ACETION BY 5 '-3' EXONLECTASE ACTIVITY OF POLYMERS ACTING ONJAADBRIDIIDE OLUCLETIDES (NUCLEIC ACID DETECTION USING EXONUCLEASE ACTIVITY OF a POLYMER 5 '-3' exonucleotid ACTING on an adjacently HYBRIDIZED oligonucleotide)", issued on day 30/1, and U.S. Pat. No. 5,35MULTIMEDIA POLYMER FOR DETECTION NUCLEIC ACID (NUCLEIC ACID DETECTION OF NUCLEIC ACID) USING NUCLEIC ACID polymerase ACTIVITY system ASSAY SYSTEM (HOMOGENEOUS assay OF NUCLEIC ACID A ASSAY SYSTEM, NUCLEIC ACID system FOR detecting NUCLEIC ACIDS) ", issued, and incorporated BY NUCLEIC ACID DETECTION OF NUCLEIC ACID DETECTION system, granted 5/11/1993.

For example, in a 5 '-nuclease reaction, a target nucleic acid is contacted with a primer and a probe (e.g., a 5' -nuclease probe of the invention) under conditions in which the primer and probe hybridize to a strand of the target nucleic acid. The target nucleic acid, primers and probes are also contacted with a nucleic acid polymerase having 5 '-3' nuclease activity. A nucleic acid polymerase having 5 '-3' nuclease activity is capable of cleaving a probe that hybridizes to a target nucleic acid downstream of a primer. The 3' end of the primer provides an initial binding site for the polymerase. The bound polymerase cleaves fragments from the probe when it encounters the 5' end of the probe. This process is also shown in fig. 2. As shown, during extension of the primer 200, the polymerase 202 cleaves the 5 '-nuclease probe 204 annealed to the target or template 206, releasing the substantially non-fluorescent donor moiety (D)208 and the acceptor moiety (a)210 from the remainder of the 5' -nuclease probe 204, which includes the quencher moiety (Q) 212. Prior to cleavage, quencher moiety (Q)212 quenches fluorescent emission from acceptor moiety (a)210, whereas, as shown, after this cleavage process, quencher moiety (Q)212 no longer quenches fluorescent emission from acceptor moiety (a) 210. In another exemplary embodiment, the 5' -nuclease probe comprises only substantially non-fluorescent donor and acceptor moieties. In these embodiments, the intensity of the fluorescent emission from the acceptor moiety will generally decrease after cleavage, as the donor and acceptor moieties are separated from each other.

The primer and probe can be designed such that they anneal in close proximity on the target nucleic acid such that binding of the nucleic acid polymerase to the 3 '-end of the primer allows it to contact the 5' -end of the probe without primer extension. The term "polymerization-independent cleavage" refers to this process. Alternatively, if the primer and probe anneal to remote regions of the target nucleic acid, respectively, polymerization will typically occur before the nucleic acid polymerase encounters the 5' -end of the probe. As polymerization continues, the polymerase constantly cleaves fragments from the 5' -end of the probe. This cleavage proceeds until the remainder of the probe has destabilized to the point where it is detached from the template molecule. The term "polymerization-dependent cleavage" refers to this process.

One advantage of polymerization-independent cleavage is that the need for nucleic acid amplification is eliminated. If the primers and probes are bound adjacently to the nucleic acid, successive rounds of probe annealing and fragment cleavage can be performed. Thus, a sufficient amount of fragments can be produced such that detection is possible also in the absence of polymerization.

In either process, a sample is provided that is believed to contain the target nucleic acid. The target nucleic acid contained in the sample may be first reverse transcribed into cDNA if desired, and then denatured by any suitable denaturing method, including physical, chemical, or enzymatic methods well known to those skilled in the art. One exemplary physical method of achieving strand separation involves heating the nucleic acids to complete (> 99%) denaturation. Typical thermal denaturation involves a temperature of about 85 ℃ to about 105 ℃ and a time of about 1 to about 10 minutes. As an alternative to denaturation, the nucleic acid may be present in the sample in single stranded form, such as when the sample comprises single stranded RNA or DNA virus.

The denatured target nucleic acid strand is typically incubated with a primer and a probe under hybridization conditions that allow the primer and probe to bind to the target nucleic acid strand. In some embodiments, two primers may be used to amplify the target nucleic acid. The two primers are typically selected such that their relative positions along the target nucleic acid are such that the extension product synthesized from one primer, when separated from its template (complementary sequence), can act as a template for extension of the other primer to produce a copy strand of defined length.

Since the two complementary strands are generally longer than the probe or primer, the two strands have more points of contact and therefore have a greater chance of binding to each other at a given time. Thus, a high molar excess of probes and primers is typically employed to allow annealing of the primers and probes beyond annealing of the template strand. In multiplex formats, multiple probes are typically used in a single reaction vessel to detect multiple target nucleic acids simultaneously.

Primers are generally of sufficient length and complementarity such that they selectively bind to the target nucleic acid under conditions selected to allow polymerization-independent or polymerization-dependent cleavage to proceed. The exact length and composition of the primer will depend on many factors, including the temperature of the annealing reaction, the source and composition of the primer, the proximity of the probe annealing site to the primer annealing site, and the primer: probe concentration ratio. For example, depending on the complexity of the target sequence, the primer typically comprises about 15-30 nucleotides, although it may contain more or fewer nucleotides.

The probe is typically annealed to its complementary target nucleic acid before the nucleic acid polymerase encounters this region of the target nucleic acid, allowing the 5 '-3' nuclease activity of the enzyme to cleave fragments from the probe. To increase the likelihood that the probe will anneal to the target nucleic acid before the polymerase reaches this hybridization region, a variety of techniques may be employed. For example, short primers generally require lower temperatures to form sufficiently stable hybridization complexes with nucleic acids. Thus, the probe can be designed to be longer than the primer, such that the probe preferentially anneals to the target nucleic acid at a higher temperature relative to primer annealing. By way of further example, the nucleotide composition of the probe may be selected to have a greater G/C content and thus greater thermal stability than the primer. Optionally, modified nucleotides can be incorporated into the introduction or probe to achieve greater or lesser thermal stability compared to a primer or probe having only unmodified nucleotides. Exemplary modified nucleotides are described further above. Thermal cycling parameters can also be varied to make use of the probeAnd differential thermal stability of the primers. For example, after a thermal cycling denaturation step, an intermediate temperature may be introduced that allows probe binding to occur but not primer binding. Thereafter, the temperature may be further reduced to allow primer annealing to occur. To favor probe binding over primer binding, a high molar excess of probe compared to primer concentration may also be used. Such probe concentrations are generally in the range of about 2 to about 20 times higher than the respective primer concentrations, and are generally about 0.5-5X 10^-7M。

Template-dependent extension of a primer is typically catalyzed by a biocatalyst (e.g., a polymerase) that catalyzes nucleotide incorporation in the presence of sufficient quantities of four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP) or analogs in a reaction mixture that additionally includes appropriate salts, metal cations, and buffers. The reaction mixture is further described above. Suitable biocatalysts that catalyze nucleotide incorporation are enzymes known to catalyze primer and template dependent DNA synthesis and have 5 '-3' nuclease activity. Exemplary DNA polymerases of this type include E.coli DNA polymerase I, Tth DNA polymerase, Bacillus stearothermophilus DNA polymerase, Taq DNA polymerase, Thermus sp ZO5 DNA polymerase, Thermotoga maritima (Thermotoga maritima) DNA polymerase, Thermotoga neapolina (Thermotoga neocolitana) DNA polymerase, and Thermotoga africana (Thermosiphora africana) DNA polymerase. The reaction conditions required for the catalytic synthesis of DNA using these DNA polymerases are well known in the art. Typically, a biocatalyst that catalyzes nucleotide incorporation is effective to cleave the probe to release the labeled fragment such that a detectable signal is generated, either directly or indirectly.

The synthesized product is typically a duplex molecule that includes a template strand and a primer extension strand. A byproduct of this synthesis is probe fragments, which may include a mixture of single nucleotide fragments, dinucleotide fragments, and larger nucleotide fragments. Repeated cycles of denaturation, probe and primer annealing, and primer extension and probe cleavage result in exponential accumulation of the region defined by the primer and exponential generation of the labeled fragment. Sufficient cycles are run to obtain detectable amounts of probe fragments, which are typically several orders of magnitude greater than the background signal. The use of a donor moiety as described herein is effective to reduce the number of cycling runs before detectable signal is generated, relative to assays that do not reduce these background signals.

In certain embodiments, the PCR reaction is performed as an automated process employing thermostable enzymes. In this process, the reaction mixture is cycled through a denaturation step, a probe and primer annealing step, and a synthesis step, in which cleavage and displacement are performed simultaneously with primer-dependent template extension. In some embodiments, the methods described herein are performed with a system. Such a system is described in more detail below. Optionally, a thermal cycler designed for use with thermostable enzymes may be employed, such as those commercially available, for example, from Applied Biosystems (Foster City, CA, USA).

Thermostable polymerases are commonly used in automated processes that effect denaturation of double-stranded extension products by exposing them to high temperatures (e.g., about 95 ℃) during PCR cycles. Representative thermostable ENZYMEs isolated from Thermus aquaticus are disclosed, for example, in U.S. Pat. No. 4,889,818 to Gelfand et al, entitled "PURIFIEDTHERMOSTABLE ENZYME", granted 12/26 1989. Additional representative thermostable polymerases include, for example, polymerases extracted from the following thermostable bacteria: thermus flavus, Thermus rubrus (Thermus rubber), Thermus thermophilus (Thermus thermophilus), Bacillus stearothermophilus (Bacillus stearothermophilus) with an optimum temperature slightly lower than those listed, Thermus lactis, Thermus rubens, Thermotoga maritima (Thermotogaritima), Theragra neapolitana (Thermato neophyllatana), Thermotoga africana (Thermosiphora africana), Thermococcus littoralis and Thermomethane thermophilus (Methanothermus thermophilus).

Hybridization of the probe to the target nucleic acid can be achieved by selecting appropriate hybridization conditions. The probe is typically: the stability of the target nucleic acid hybrid is selected to be consistent with assay and wash conditions such that a stable detectable hybrid is formed only between the probe and the target nucleic acid. Manipulation of one or more of the different assay parameters determines the exact sensitivity and specificity of a particular hybridization assay.

More specifically, hybridization between complementary bases of nucleic acids (e.g., DNA, RNA, PNA, or combinations thereof) occurs under a wide variety of conditions that vary in temperature, salt concentration, electrostatic strength, and buffer composition. Examples of such conditions and methods of their use are described for example in Tiissen,Hybridization with Nucleic Acid Probesvol.24, Elsevier Science (1993) and Hames and Higgins, supra. Hybridization is typically carried out between about 0 ℃ to about 70 ℃ for a period of about 1 minute to about 1 hour, depending on the nature of the sequence to be hybridized and its length. However, it will be appreciated that hybridization may occur within seconds or hours, depending on the conditions of the reaction. For example, typical hybridization conditions for a mixture of two 20-mer sequences are to warm the mixture to 68 ℃ and then cool it to room temperature (22 ℃) for 5 minutes, or at a very low temperature such as 2 ℃. Hybridization between nucleic acids can be performed using buffers such as Tris-EDTA (TE), Tris-HCl and HEPES, salt solutions (e.g. NaCl, KCl, MgCl₂) Or other aqueous solutions, reagents, and chemicals. Examples of such agents include single-stranded binding proteins such as Rec A protein, T4 gene 32 protein, E.coli single-stranded binding proteins, and nucleic acid major and minor groove binding proteins. Other examples of such agents and chemicals include divalent ions, multivalent ions, and intercalating substances (intercalling substance) such as ethidium bromide, actinomycin D, psoralen, and angelicin.

Almost any method for detecting a target nucleic acid can be used in the present invention. Commonly used methods include real-time amplification detection with 5' -nuclease probes, hybridization probes, or hairpin probes (e.g., molecular beacons), detection of labels incorporated into the amplification primers or the amplified nucleic acids themselves after, for example, electrophoretic separation of the amplification products from unincorporated labels, hybridization-based assays (e.g., array-based assays), and/or detection of secondary reagents (secondary reagents) that bind nucleotides. These general methods are also described, for example, in Sambrook and Ausubel 1 and 2, supra.

Hairpin probes, such as molecular beacons, are oligonucleotides designed to perform real-time detection and quantification of target nucleic acids. The 5 'and 3' ends of hairpin probes typically comprise a labeling moiety that confers the detectable properties of the probe. In exemplary embodiments, one of the termini is linked to a substantially non-fluorescent donor moiety and to an acceptor moiety (e.g., a fluorescent dye), and the other terminus is linked to a quencher moiety capable of quenching fluorescent emission from the acceptor moiety. When the hairpin probe is free in solution, i.e., not hybridized to a second nucleic acid, the stem region of the probe is stabilized by complementary base pairing. This self-complementary pairing results in a probe "hairpin loop" structure in which the acceptor and quencher moieties are in close proximity to one another. In this conformation, the quencher moiety will quench the acceptor moiety. The loop of the hairpin probe typically comprises a sequence complementary to the sequence to be detected in the target nucleic acid, such that hybridization of the loop to its complementary sequence in the target forces the stem region to dissociate, thereby distancing the acceptor and quencher moieties from each other. This leaves the acceptor moiety unquenched, resulting in increased fluorescence from the hairpin probe.

By way of further example, fig. 3 shows a hairpin probe 300 comprising a substantially non-fluorescent donor moiety (D)302, an acceptor moiety (a)304, and a quencher moiety (Q) 306. As shown, quencher moiety (Q)306 will quench the fluorescent emission from acceptor moiety (a)304 when hairpin probe 300 is free in solution, but will not when hairpin probe 300 is hybridized to target nucleic acid 308. In another representative embodiment, a hairpin probe includes only substantially non-fluorescent donor and acceptor moieties. In these embodiments, the intensity of fluorescence from the acceptor moiety will generally decrease when the probe hybridizes to the target nucleic acid due to its conformational change.

Details regarding standard methods of making and using hairpin probes are generally well known to those skilled in the art and are also described, for example, in the following references: leone et al (1995)"molecular probes combined with amplification by NASBA enabled homogenetic real-time detection of RNA (homologous real-time detection of RNA in combination with NASBA amplification)",Nucleic Acids Res.26: 2150-: spectral genotyping of humanallels (molecular beacons: spectral genotyping of human alleles) ",Science279: 1228-1229, Fang et al, (1999) "design a novel molecular beacon for surface-immobilized DNA hybridization sessions",J.Am.Chem.Soc.121: 2921-2922 and Marras et al, (1999) "multiple detection of single-nucleotide variation using molecular beacons",Genet.Anal.Biomol.Eng.14: 151-156. There are a number of commercial suppliers of standard and custom hairpin probes that can be used in the methods described herein, including Oswel Research products Ltd. (UK), Research Genetics (Invitrogen, Huntsville, AL, a division of the USA), and Midland verified Reagent Company (Midland, TX, USA). There are a number of kits which employ hairpin probes, such as Sentinel from Stratagene (La Jolla, CA, USA)^TMMolecular Beacon Allelic diagnosis Kits and various Kits of Eurogentec SA (Belgium) and Isogen bioscience BV (Netherlands). These kits are also optionally suitable for use in the methods described herein.

Hybridization probes typically function in pairs and can be used to achieve various types of real-time target nucleic acid detection, including quantitation, mutation detection, melting temperature (T)_m) Multiplexing (multiplexing) and colorimetric multiplexing (color multiplexing). Aspects of certain hybridization probe assays are described, for example, in Brega et al, (2004) "Real-time PCR for dihydrofolate reductase single-nucleotide polymorphisms in Plasmodium vivaxoides (Real-time PCR for detection of single nucleotide polymorphisms of the dihydrofolate reductase gene in Plasmodium vivaxolate)",Antimicrob Agents Chemother.48(7): 2581-2587, Perell et al, (2004) "A LightCycler real-time PCRhybridization probe assay for detecting food-borne thermophilicCamphyllobacter," real-time PCR hybridization Probe assay for detecting food-infectious thermophilus Campylobacter),Mol Cell Probes.18(5): 321-327 and Whiley et al, (2003) "Detection of Neisseria Meningitidis in clinical samples by dual real-time PCR targeting the porA and ctrA genes",Mol Diagn.7(3-4)：141-145。

hybridization probe assays typically involve hybridizing a pair of labeled probes to a target or template nucleic acid in sufficient proximity to one another that energy transfer occurs between the labeled moieties. More specifically, one hybridization probe of a hybridization probe pair (the "donor probe") typically includes at least one substantially non-fluorescent donor moiety (e.g., 5-or 6-carboxy Dmf), while the other hybridization probe (the "acceptor probe") includes at least one acceptor moiety (e.g., LC-Red 610, LC-Red 640, LC-Red 670, LC-Red 705, JA-270, CY5, or CY 5.5). By way of further example, fig. 4 shows that when a donor probe 400 having a substantially non-fluorescent donor moiety (D)402 and an acceptor probe 404 having an acceptor moiety (a)406 are free in solution, non-fluorescent excitation energy is not transferred from the substantially non-fluorescent donor moiety (D)402 to the acceptor moiety (a) 406. Conversely, when both the donor probe 400 and the acceptor probe 404 hybridize to the target or template nucleic acid 408, the acceptor moiety (a)406 receives non-fluorescent excitation energy transferred from the substantially non-fluorescent donor moiety (D)402 and in response fluoresces, the 402 being sufficiently close to the 406 on the target or template nucleic acid 408. In another exemplary embodiment, one hybridization probe of a hybridization probe pair includes both a substantially non-fluorescent donor moiety and an acceptor moiety, while the other hybridization probe includes a quencher moiety. In this embodiment, when the probe pair hybridizes to the target nucleic acid, the detectable fluorescence emission from the acceptor moiety is reduced and the primer quencher moiety quenches the fluorescenceIt is used. Fluorescence emitted by the acceptor moiety of the hybridization probe can be detected by various known methods, including the use of LightCyclersThose of the system (Roche Diagnostics Corporation, Indianapolis, IN, USA).

In other illustrative embodiments using the biomolecules described herein, labeled primers are used to achieve real-time target nucleic acid detection. For example, fig. 5 shows a primer 500 comprising a substantially non-fluorescent donor moiety (D)502, an acceptor moiety (a)504, and a quencher moiety (Q) 506. Prior to primer extension, primer 500 forms a hairpin loop structure such that quencher moiety (Q)506 quenches the fluorescence emitted by acceptor moiety (a) 504. Upon extension of primer 500 (binding to template nucleic acid 508), the hairpin loop structure dissociates and hybridizes to the newly formed complementary sequence, separating acceptor moiety (a)504 from quencher moiety (Q)506, such that fluorescent emission from acceptor moiety (a)504 can be detected. In other embodiments, the primer comprises only substantially non-fluorescent donor moieties and acceptor moieties. Primer-based Real-time target nucleic acid detection methods that may be suitable for biomolecules of the invention are also described, for example, in Huang et al, (2004) "Real-time assay of transcriptional activity using the duplex scorpion primer" (Real-time quantitative determination of telomerase activity using a double-stranded scorpion primer) ",Biotechnol Lett.26(11): 891-895, Asselbergs et al, (2003) "Rapid detection of apoptosis in aqueous fresh-time reverse transcriptase polymerase chain reaction measurement of the small cytoplasmic RNA Y1 (Rapid detection of apoptosis by real-time reverse transcriptase polymerase chain reaction of small cytoplasmic RNA Y1)",Anal Biochem.318(2): 221-229 and Nuoovo et al, (1999) "In situ amplification using universal energy transfer-labeled primers",J Histochem Cytochem.47(3)：273-280。

another exemplary application of certain embodiments of the biomolecules described herein is for nucleic acid sequencing. In general, nucleic acid sequencing protocols involve extension/termination reactions of primer nucleic acids. The reaction mixture typically includes deoxynucleoside triphosphates (dNTPs) and a polymerase enzyme, which are used to extend the primer. In addition, the reaction mixture also includes at least one dideoxynucleoside triphosphate (ddNTP) or other terminator nucleotide, which when incorporated onto the extended primer prevents further extension of the primer. After termination of the extension reaction, the different termination products formed can be separated and analyzed to determine the base sequence of the target nucleic acid.

Nucleic acid sequencing is generally divided into two categories: "dye primer sequencing" and "dye terminator sequencing". In dye primer sequencing, a fluorescent dye (e.g., an acceptor moiety and a substantially non-fluorescent donor moiety as described herein paired) is incorporated onto the primer being extended. Four separate extension/termination reactions were then run in parallel, each extension reaction containing a different termination nucleotide to terminate the extension reaction. After termination, the reaction products are usually separated by gel electrophoresis and analyzed (Ansorge et al (1987)Nucleic Acids Res.15：4593-4602)。

In one variation of dye primer sequencing, different primers are used in four separate extension/termination reactions, each primer containing a different spectrally resolvable dye. After termination, the reaction products of the four extension/termination reactions are typically pooled, electrophoretically separated and detected in a single lane (Smith et al, (1986)Nature321: 674-679). Thus, in this variation of dye primer sequencing, products from more than one extension/termination reaction can be detected simultaneously by using primers containing a set of spectrally resolvable dyes.

In dye terminator sequencing, a fluorescent dye (e.g., an acceptor moiety and a substantially non-fluorescent donor moiety as described herein paired) is attached to each terminator nucleotide (e.g., a 2' -terminator or a dideoxynucleotide triphosphate). An extension/termination reaction is then performed in which the primer is extended with deoxynucleotide triphosphates until a labeled termination nucleotide is incorporated into the extended primer, preventing further extension of the primer. Once the reaction is terminated, the reaction product of each of the terminated nucleotides is isolated and detected. In one embodiment, a separate extension/termination reaction is performed for each of the four terminator nucleotides. In another embodiment, a single extension/termination reaction is performed that contains four terminating nucleotides, each labeled with a different spectrally resolvable fluorescent dye as described herein.

Thus, according to one aspect of the invention, there is provided a method of dye primer sequencing using one or more oligonucleotide reagents of the invention. According to this method, a mixture of extended labeled primers is formed by hybridizing a target nucleic acid to a labeled primer in the presence of deoxynucleotide triphosphates, at least one terminator nucleotide, and a polymerase. The labeled primer includes an oligonucleotide sequence that is complementary to a portion of the target nucleic acid sequence being sequenced, and an energy transfer fluorescent dye attached to the oligonucleotide (e.g., an acceptor moiety and a substantially non-fluorescent donor moiety as described herein paired).

According to this exemplary method, a polymerase extends a primer with deoxynucleotide triphosphates until a terminator nucleotide is incorporated that can terminate extension of the primer. Following termination, the extended primers in the mixture are typically separated (e.g., electrophoretically and/or chromatographically separated). The sequence of the target nucleic acid is then determined by detecting the extended primer.

In another exemplary embodiment of this method, four dye primer sequencing reactions are run, each primer sequencing reaction comprising a differently labeled primer and a different terminator nucleotide (e.g., ddATP, ddCTP, ddGTP, and ddTTP). After running four dye primer sequencing reactions, the resulting mixture of extended primers can be pooled. The extended primers in the mixture can then be separated and the fluorescent signal from each of the four differently labeled primers detected to determine the sequence of the target nucleic acid.

According to another embodiment of the invention, there is provided a method of dye terminator sequencing with one or more terminator nucleotides (e.g., 2' -terminator nucleotides or dideoxynucleotide triphosphates) labeled with an energy transfer dye of the invention (e.g., an acceptor moiety and a substantially non-fluorescent donor moiety as described herein in a pair). According to this method, a mixture of extended primers is formed by hybridizing a target nucleic acid to a primer in the presence of deoxynucleotide triphosphates, at least one labeled stop nucleotide, and a polymerase. In some embodiments of this method, the step of forming a mixture of extended primers comprises hybridizing the target nucleic acid with four differently labeled terminator nucleotides. The polymerase extends the primer with deoxynucleoside triphosphates until a labeled stop nucleotide is incorporated into the extended primer. After termination, the extension primers in the mixture are typically isolated. The sequence of the target nucleic acid is then determined by detecting fluorescence from the labeled stop nucleotide incorporated into the extended primer.

One exemplary use of the biomolecules of the present invention includes the analysis of proteins. For example, fig. 6 shows assays that can be used to assess response to protein cleavage. As shown, certain amino acids 602 of protein 600 include a substantially non-fluorescent donor moiety (D)604, an acceptor moiety (a)606, or a quencher moiety (Q) 608. Prior to cleavage (e.g., by protease cleavage and/or chemical cleavage), quencher moiety (Q)608 quenches the fluorescent emission from acceptor moiety (a)606, which 606 receives excitation energy transferred from substantially non-fluorescent donor moiety (D) 604. After cleavage, the quencher moiety (Q)608 no longer quenches this fluorescence, which can be detected by a variety of methods well known to those skilled in the art. In an exemplary variation of this method, the protein labels only the substantially non-fluorescent donor and acceptor moieties, such that upon cleavage the intensity of fluorescence emitted from the acceptor moiety is reduced. Various aspects of protease cleavage assays applicable to the biomolecules of the invention are described, for example, in Jenny et al, (2003) "A clinical review of the methods for cleavage of fusion proteins with thrombin and factor Xa (for cleavage of fusion proteins with thrombin and factor Xa)Review of the method of (1) ",Protein Expr Purif.31(1): 1-11 and Funovics et al, (2003) "Protease sensors for bioimaging",Anal Bioanal Chem.377(6)：956-963。

other representative proximity assays that may be performed with certain biomolecules of the invention include lipid mixing assays based on energy transfer. For example, fig. 7 illustrates certain steps performed in a lipid mixing assay according to one embodiment of the present invention. As shown, labeled membrane 700 includes a donor lipid 702 and an acceptor lipid 706, the 702 including a substantially non-fluorescent donor moiety (D)704, the 706 including an acceptor moiety (a) 708. Prior to fusing the labeled membrane 700 with the unlabeled membrane 710, the acceptor moiety (a)708 absorbs the excitation energy transferred from the substantially non-fluorescent donor moiety (D)704 and in response fluoresces, the 704 being sufficiently proximal to the acceptor moiety (a)708 in the labeled 700. This fluorescence decreases once fusion of the two membranes occurs as the distance between the donor lipid 702 and the acceptor lipid 706 increases.

Methods for detecting fluorescence from acceptor moieties in these and other proximity assays are well known to those skilled in the art. Some of these detection methods and related systems, including the use of photomultiplier tubes or charge-coupled devices, are described further below.

In other representative embodiments, the present invention provides methods of conducting business involving the biomolecules described herein. For example, FIG. 8 is a block diagram illustrating certain steps performed in a business method in accordance with one embodiment of the present invention. As shown in step 800, the method includes receiving a purchase order from a customer for at least one biomolecule described herein. In addition, the method includes supplying the biomolecule to the customer in accordance with the purchase order (step 802). For example, in some embodiments, the business entity receives the purchase order through direct submission by the customer or an agent thereof, through a postal or other delivery service (e.g., a general postman), through telephone communication, through email communication or other electronic media, or any other suitable method. In some embodiments, the ordered and/or supplied biomolecules are included in a kit as described herein. In addition, the biomolecules are supplied or provided to the customer by any suitable method (e.g., in exchange for some form of payment), including direct delivery by the customer or an agent thereof, by postal or other delivery service such as a general postman.

VII. System

The invention also provides a system for detecting energy emission from a acceptor moiety in response to non-fluorescent energy transferred from a substantially non-fluorescent donor moiety. These systems can be used to perform many different assays, including the proximity assays mentioned herein. These systems include one or more biomolecules that are labeled with at least one or more substantially non-fluorescent donor moieties. In certain embodiments, the biomolecules are arrayed on a solid support, while in other embodiments they are provided in one or more containers, e.g., for assays performed in solution. These systems also include a radiation source and at least one detector or detection device (e.g., a spectrometer) capable of at least detecting energy emitted from the receiver portion when the receiver portion is sufficiently proximate to the substantially non-fluorescent donor portion. In addition, these systems optionally further comprise at least one thermal regulator (e.g., a thermal cycling device) operably connected to the container or solid support to regulate the temperature in or on the container and/or at least one fluid transfer device (e.g., an automated pipettor) capable of transferring fluid to and/or from the container or solid support, e.g., to perform one or more proximity assays in or on the solid support.

The detector is typically configured to detect a detectable signal, e.g., generated in or proximate to another device of a given assay system (e.g., in the container and/or on the solid support). Suitable signal detectors optionally used or adapted for use in the present invention are capable of detecting, for example, fluorescence, phosphorescence, radioactivity, absorbance, refractive index, luminescence, orAnd (4) quality. The detector optionally monitors one or more signals from, for example, upstream and/or downstream of a given assay step being performed. For example, the detector optionally monitors a plurality of optical signals corresponding in position to "real-time" results. Examples of detectors or sensors include photomultiplier tubes, CCD arrays, optical sensors, temperature sensors, pressure sensors, pH sensors, conductivity sensors, or scanning detectors. More specific exemplary calibration detectors optionally used to perform the methods of the invention include, for example, resonance light scattering detectors, emission spectroscopes, fluorescence spectroscopes, phosphorescence spectroscopes, luminescence spectroscopes, spectrophotometers, and photometers. Detectors are also described, for example, in the following documents: skoog and the like, and,Principles of Instrumental Analysis5 th edition, Harcourt Brane College Publishers (1998), Currell,Analytical Instrumentation：Performance Characteristics and Quality，John Wiley &sons, inc. (2000), Sharma et al,Introduction to Fluorescence Spectroscopy，John Wiley & Sons，Inc.(1999)，Valeur，Molecular Fluorescence：Principles and Applications，John Wiley &sons, inc. (2002) and Gore,Spectrophotometry and Spectrofluorimetry：A Practical Approach2 nd edition, Oxford University Press (2000).

The systems of the present invention also typically include a controller operatively connected to one or more devices of the system (e.g., the detector, the thermal regulator, and/or the fluid transfer device) to control the operation of the devices. More specifically, a controller is typically included in the system as a stand-alone or built-in system component for, for example, receiving data from the detector, effecting and/or regulating the temperature in the vessel, effecting and/or regulating the flow of fluid to or from a selected vessel. The controller and/or other system components are optionally coupled to a suitably programmed processor, computer, digital device, information appliance, or other logic device (e.g., including an analog-to-digital or digital-to-analog converter as desired) that functions to direct the operation of such components in accordance with preprogrammed or user-entered instructions, to receive data and information from such components, and to parse, manipulate, and report such information to the user. Suitable controllers are known in the art and are available from a variety of commercial sources.

Any controller or computer optionally includes a monitor, which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., an active matrix liquid crystal display or a liquid crystal display), or other display. Computer circuitry is often placed in a box that includes numerous integrated circuit chips, such as microprocessors, memory, interface circuits, and other circuit chips. The case also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writable CD-ROM, and other common peripheral components. An input device such as a keyboard or mouse optionally provides input operations for the user. These devices are further described below.

The computer typically includes appropriate software to receive user instructions in the form of user input into a set of parameter fields (e.g., in a GUI), or in the form of pre-programmed instructions (e.g., pre-programmed for a variety of different specific operations). The software will then translate these instructions into the appropriate language to direct the operation of the controller or controllers to perform the desired operations. The computer then receives data from sensors/detectors included in the system, for example, and interprets the data, provides it in a form understandable to the user, or uses it to initiate further controller instructions in accordance with a programmed program, for example, to control a fluid flow regulator in response to fluid weight data received from a scale.

FIG. 9 is a schematic diagram illustrating a representative system including logic devices in which aspects of the present invention may be embodied. Those skilled in the art and guided by the teachings herein provided will appreciate that the present invention optionally may be implemented in hardware and/or software. In some embodiments, the different aspects of the invention are performed in client-side logic circuitry or server-side logic circuitry. It will be appreciated in the art that the invention or a device thereof may be embodied in a media program component (e.g., a fixed media component) containing logic instructions and/or data that, when loaded into an appropriately configured computer apparatus, cause the apparatus to perform as required. It will also be appreciated that the fixed media containing the logic instructions may be delivered to an observer of the fixed media for physical loading into the observer's computer, or the fixed media containing the logic instructions may be stored on a remote server that the observer accesses through a communications medium to download the program components.

More specifically, FIG. 9 shows a computer 900 to which a detector 902 (e.g., a spectrometer such as a spectrofluorimeter), a fluid transfer device 904, and a thermal regulator 908 are operatively connected. Optionally, one or more of these devices may be operatively coupled to computer 900 by a server (not shown in FIG. 9). During operation, the fluid transfer device 904 typically transfers the reaction mixture or components thereof to the porous container 906. Thermal conditioning (e.g., thermal cycling) is typically achieved by thermal conditioner 908 which is in thermal communication with porous container 906. The detector 902 typically detects a detectable signal (e.g., fluorescence emission) generated before, during, and/or after a given proximity assay is performed in the system. Those skilled in the art will recognize that one or more of the components of the system shown in fig. 9 are optionally made integral with each other (e.g., in the same housing).

VIII. kit

The reaction mixture or components thereof (e.g., biomolecules) employed in the methods of the invention are optionally packaged in a kit. In certain embodiments, spectrally resolvable acceptor moieties are labeled on different biomolecules included in the kits, e.g., to perform certain multiplex applications (multiplexing applications) using these kits. In some embodiments of these kits, only one type of suitable substantially non-fluorescent donor moiety is included, such that only a single energy source is required to excite each of the different acceptor moieties.

In addition, the kit may also include suitable packaged reagents and materials to perform real-time monitoring of biopolymer synthesis, nucleic acid sequencing reactions, nucleic acid amplification or other proximity assays, such as buffers, enzymes, standards or controls, salts, metal ions, primers, probes, extendable nucleotides, terminating nucleotides, glycerol, dimethyl sulfoxide, and/or poly rA, as well as instructions for performing a particular process or assay. In some of these embodiments, the kit further comprises at least one pyrophosphatase (e.g., a thermostable pyrophosphatase), e.g., for minimizing pyrophosphorolysis, and/or uracil N-glycosylase (UNG) and optionally dUTP, e.g., for applications where it is desirable to prevent carryover contamination from occurring. The kit components, such as biomolecules, are typically provided in one or more containers.

By way of further example, the kits of the present invention include many different embodiments for performing a variety of assay and/or synthesis reactions. Some of these applications are also described above. For example, in some embodiments, the kit includes an immunoglobulin labeled as described herein and instructions for detecting binding of the immunoglobulin to a target epitope as part of a given immunoassay. Optionally, the kit includes a labeled lipid and instructions for using the lipid to perform a particular proximity assay, such as a membrane fusion assay. In certain embodiments, the kit comprises a primer and instructions for extending the primer, e.g., in a nucleic acid sequencing reaction or a nucleic acid amplification reaction. In these embodiments, the primers and/or the optionally included extendable nucleotides, terminating nucleotides (e.g., dideoxycytidine triphosphate, dideoxynucleotide triphosphate, and dideoxynucleotide triphosphate) and/or probes (e.g., hybridization probes, 5' -nuclease probes, and hairpin probes) are typically labeled as described herein. In some other exemplary embodiments, the kit includes biopolymer synthesis reagents (e.g., polypeptide synthesis reagents or nucleic acid synthesis reagents) and instructions for using these reagents to synthesize a biopolymer.

IX. example

It should be recognized that the examples and embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention as claimed. It should also be appreciated that various modifications or changes may occur to those skilled in the art from the examples and embodiments described herein, which modifications or changes are also encompassed within the spirit and purview of this application and scope of the appended claims.

Example I: comparison of the detected fluorescence

This example shows the fluorescence emissions detected from an oligonucleotide labeled with a fluorescent moiety and an oligonucleotide labeled with a substantially non-fluorescent donor moiety in different samples, respectively. Specifically, FIG. 10 is a graph of overlapping spectra obtained from excitation and emission scans of oligonucleotides labeled with 6-carboxyfluorescein (6-FAM), 4 ', 5' -dimethoxy-5-carboxyfluorescein (5-Dmf), or 4 ', 5' -dimethoxy-6-carboxyfluorescein (6-Dmf). The ordinate of the graph shown in fig. 10 represents absolute fluorescence, and the abscissa represents wavelength (nm). As shown in the figure, the curve of the excitation scan of the 6-FAM-labeled oligonucleotide obtained at 515nm is denoted by "6-FAM ex", and the curve of the emission scan of the 6-FAM-labeled oligonucleotide obtained at 493nm is denoted by "6-FAM em". The curve of the excitation scan of the 5-Dmf-labeled oligonucleotide obtained at 545nm is indicated with "5-Dmf ex", while the curve of the emission scan of the 5-Dmf-labeled oligonucleotide obtained at 520nm is indicated with "5-Dmf em". The curve of the excitation scan of the 6-DmF-labelled oligonucleotide obtained at 545nm is indicated with "6-DmF ex", while the curve of the emission scan of the 6-DmF-labelled oligonucleotide obtained at 520nm is indicated with "6-DmF em". By way of further example, FIG. 11 is an abstract view of a detail of the graph shown in FIG. 10, showing overlapping spectra obtained from excitation and emission scans of 5-Dmf and 6-Dmf labeled oligonucleotides, respectively. The concentration of labeled nucleotides in each of the individual scan samples was 0.15. mu.M.

Example II: synthesis of biopolymer synthesis reagents

This example describes representative synthetic pathways for biopolymer synthesis reagents comprising substantially non-fluorescent donor moieties. Specifically, the synthesis scheme of 5-and 6-dimethoxyfluorescein-DMT-CX-linker-phosphoramidite according to one embodiment of the present invention is described in this example. As a summary, fig. 12 illustrates some of the steps in this synthesis scheme.

Synthesis of 5 and 6-dimethoxyfluorescein (Dmf) isomers

The synthesis of DmF was performed according to the procedure of the following patents: khanna et al, U.S. Pat. No. 4,318,846 entitled "NOVEL ETHER SUBSTITUTED FLUORESCENTERINE POLYAMINO ACID COMPOSITS AS FLUORESCERS ANDQUENCHERS (NOVEL Ether-SUBSTITUTED fluorescein polyamino ACID COMPOUNDS AS FLUORESCERS and quenchers)" was granted 3/9 in 1982. After the post-treatment, the product is purified by column chromatography. As shown in fig. 13 (ordinate represents absorbance units, abscissa represents retention time (min)), HPLC analysis of purified DmF showed two peaks due to positional isomers, which was expected. These positional isomers were assigned to 5-Dmf and 6-Dmf. In addition, the identity (identity) of these compounds was confirmed by mass spectrometry data.

Pivaloylation of the 5-and 6-Dmf isomers

To a solution of Dmf (200mg, 0.46mmol) in pyridine (5ml) was added N, N-diisopropylethylamine (3.0 ml). The reaction mixture was then cooled in a water bath pre-cooled with ice for 20 minutes. To this reaction mixture was added pivaloyl chloride (500mg, 4.15mmol) dropwise. After the addition, the reaction mixture was stirred for a further 15 minutes under cooling. The cooling bath was then removed and the reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and then purified by column chromatography. See fig. 14, which is an HPLC trace showing detection of pivaloyl 5-DmF and pivaloyl 6-DmF (ordinate represents absorbance units, abscissa represents retention time (min)). The identity of these compounds was confirmed by mass spectrometry.

pivaloyl-5-and-6-Dmf-DMT-CX-linker

HBTU (3ml) was added to a solution of pivaloyl Dmf (220mg) and DMT-CX-linker (195mg, prepared in Roche, Penzberg) in DMSO (2ml), dimethylformamide (3ml) under cooling. After about 5 minutes, diisopropylethylamine (0.4ml) was added and stirring was continued for 3 hours under cooling (0-5 ℃ C., bath temperature). As shown in fig. 15 (ordinate represents absorbance unit, abscissa represents retention time (min)), no raw material was observed after 3 hours. The solvents (DMF and DMSO) in the reaction mixture were removed under high vacuum. The product was purified by column chromatography (see HPLC chromatogram of fig. 16 (ordinate represents absorbance unit, abscissa represents retention time (min)), and confirmed by mass spectrometry analysis the isolation yield was 84% (320 mg).

5 and 6-Dmf-DMT-CX-linker-phosphoramidites

To a solution of pivaloyl-Dmf-DMT-CX-linker (100mg, 0.095mmol) in dichloromethane (6ml) was added diisopropylethylamine (300mg, 0.84mmol) at room temperature, and the resulting reaction mixture was cooled to 5 ℃ in ice-cold water (bath temperature). Cyanoethyldiisopropylphosphoramidite reagent (28mg, 0.12mmol) was added to the reaction mixture and stirring was continued at 5 ℃. After 1 hour, cyanoethyldiisopropylphosphoramidite chloride reagent (28mg, 0.12mmol) was added and the reaction mixture was left at 0 ℃ for a further 16 hours. Figure 17 is an HPLC trace showing detection of product after this 16 hour period. As shown in fig. 18 (ordinate represents absorbance units, abscissa represents retention time (min)), HPLC analysis of the reaction mixture after 18 hours showed no starting material present and a new peak appeared at 28 minutes. The product was purified by flash column chromatography (isolation yield 100mg, 80%) and confirmed by mass spectrometry and 31P NMR analysis (see fig. 19).

Chromatographic separation of dimethoxy fluorescein isomer

FIG. 20 shows certain steps in a purification scheme to separate 5-and 6-dimethoxyfluorescein isomers from each other. More specifically, the separation of the Dmf isomer was achieved by flash column chromatography using a BiotageC-18 column and 0.1M TEAA-acetonitrile as elution buffer. FIGS. 21 and 22 are chromatograms showing detection of 5-Dmf and 6-Dmf, respectively (ordinate represents absorbance units, abscissa represents retention time (min)). Fractions corresponding to 5-DmF and 6-DmF were pooled separately and lyophilized. These isomers were further confirmed by 1H NMR data for [ (5-Dmf NMR data: Δ 8.34(1H, s, Ar), 8.25(1H, d, Ar), 7.24(1H, d, Ar), 6.63(2H, d, Ar), 6.30(2H, d, Ar), 3.95(6H, s, OMe)) and 6-Dmf NMR data: δ 8.13(1H, d, Ar), 7.89(1H, d, Ar), 7.56(1H, s, Ar), 6.63(2H, d, Ar), 6.29(2H, d, Ar), 3.95(6H, s, OMe) ].

Synthesis of 6-dimethoxyfluorescein-DMT-CX-linker-phosphoramidite

The synthesis of 6-Dmf-DMT-CX-linker-phosphoramidite was the same as the synthesis of 5-and 6-Dmf-DMT-CX-linker-phosphoramidite described above. In particular, fig. 23 shows certain steps in this synthetic scheme. In addition, FIGS. 24-26 are HPLC traces showing detection of pivaloylated 6-Dmf, 6-Dmf-CX-linker, and 6-Dmf-CX-linker-phosphoramidite, respectively (ordinate represents absorbance units, abscissa represents retention time (min)).

Synthesis of 5-dimethoxyfluorescein-DMT-CX-linker-phosphoramidite

The synthesis of 5-Dmf-DMT-CX-linker-phosphoramidite is the same as the synthesis of 5-and 6-Dmf-DMT-CX-linker-phosphoramidite described above. In particular, fig. 27 shows certain steps in this synthetic scheme. In addition, FIGS. 28 and 29 are HPLC traces showing detection of pivaloylated 5-DmF and 6-DmF-CX-linker, respectively (ordinate represents absorbance units, abscissa represents retention time (min)).

Example III: hybridization probe assay

Phosphoramidites of the dye dimethoxyfluorescein (Dmf) were synthesized as described in example II and incorporated into the oligonucleotide at the 3' -end. This oligonucleotide or donor probe is labeled with LightCyclerAcceptor probes of Red 640(LC-Red 640) are hybridized together with the target oligonucleotide. Fluorescence measurements were performed in conjunction with control experiments in which a similar 6-carboxyfluorescein (6-FAM) donor probe was studied. The results showed that the donor probe with DmF had no detectable fluorescence. Specifically, when an excitation scan was performed, the fluorescence emission of the LC-Red 640 dye of the acceptor probe (at 640nm) was observed while scanning the excitation wavelength of 400-630nm, resulting in an excitation peak observed at about 520nm, which coincides with the maximum absorbance of the Dmf dye. This suggests that substantially non-fluorescent DmF transfers absorbed light to LC-Red 640 as non-fluorescent energy. For one of the isomers of the Dmf dye (i.e., 5-Dmf), the intensity of the new excitation peak is about half that of 6-FAM. FIG. 30 shows a plot of the overlapping spectra obtained from these excitation scans, where the ordinate of the graph represents absolute fluorescence and the abscissa represents wavelength (nm). As shown, the trace relating to the scan of the 6-FAM donor probe is labeled "6-FAM", while the trace relating to the scan of the Dmf donor probe is labeled "5-Dmf" and "6-Dmf". By way of further example, FIG. 31 is a plot of the overlaid spectra of FIG. 30 normalized to a fluorescence excitation scan (i.e., the ordinate of the plot represents normalized fluorescence and the abscissa represents excitation wavelength (nm)).

When an emission scan is performed, no signal is obtained from the DmF-labeled donor probe. In contrast, a large emission was detected from the 6-FAM label in the control experiment. FIG. 32 provides a plot of the overlaid spectra obtained from these scans, where the ordinate of the plot represents absolute fluorescence and the abscissa represents wavelength (nm). Specifically, the trace of the emission scan obtained at the 493nm excitation wavelength, denoted as "6-FAM", was determined from hybridization probes including 6-FAM labeled donor probes. Traces of scans obtained at 514nm excitation wavelength from assays including Dmf labeled donor probes are labeled "5-Dmf" and "6-Dmf". An assay was also performed in which no donor probe was present in the reaction mixture as a negative control. The trace of the emission scan obtained at 514nm excitation wavelength from one of these control assays is denoted "control 1" and the trace of the emission scan obtained at 493nm excitation wavelength from the other of these control assays is denoted "control 2".

Example IV: melting curve analysis

Melting curve analysis is performed with a hybridization probe pair comprising a donor probe labeled with a fluorescent moiety or a substantially non-fluorescent moiety. More specifically, one set of melting curve assays includes a hybridization probe pair in which the donor probe is labeled at the 3 '-end with 6-FAM and the acceptor probe is labeled at the 5' -end with CY 5.5. Another set of assays includes pairs of hybridization probes in which the donor probe is labeled 5-Dmf at the 3 '-end and the acceptor probe is labeled CY5.5 at the 5' -end. The same target nucleic acid was used in each assay, and the copy number was the same, i.e., 100,000 copies. FIGS. 33 and 34 show melting curves obtained from these analyses, where the ordinate of the graph represents raw (raw) fluorescence and the abscissa represents temperature (. degree.C.). The trace obtained for the assay including the 6-FAM donor probe is labeled "6-FAM" and the trace obtained for the assay including the 5-Dmf donor probe is labeled "5-Dmf". A negative control was also performed in which there was no donor probe in the reaction mixture. As shown in fig. 33 and 34, each melting curve assay was performed in duplicate.

Example V: hybridization probe based real-time PCR monitoring

The hybridization probe pairs described in example IV were also used to monitor the polymerase chain reaction. In particular, FIGS. 35 and 36 show the growth curves obtained from these analyses, where the ordinate of the graph represents the raw fluorescence and the abscissa represents the cycle number. The trace obtained for the assay including the 6-FAM donor probe is labeled "6-FAM" and the trace obtained for the assay including the 5-Dmf donor probe is labeled "5-Dmf". A negative control was also performed in which there was no donor probe in the reaction mixture. The same target nucleic acid was used in each assay, and the copy number was the same, i.e., 100,000 copies. As shown in fig. 35 and 36, each PCR was performed in duplicate.

Example VI: real-time PCR monitoring based on 5' -nuclease probes

The PCR assay is also monitored with a 5' -nuclease probe that includes a substantially non-fluorescent moiety or lacks such a moiety in the energy transfer pair. FIGS. 37A and 37B show the 5' -nuclease probes used in these assays. As shown in FIG. 37A, probe 600(Dmf probe) includes a Quencher moiety (Q) at the 5' -end of the nucleic acid, i.e., a Black Hole Quencher^TMOr BHQ^TM(Biosearch Technologies, Inc., Novato, Calif., USA). In addition, probe 600 further includes an energy transfer pair (ET pair) comprising DmF (donor moiety) and CY5.5 (acceptor moiety). The probe 602 shown in fig. 37B (non-DmF probe) is identical to probe 600, except that Fluorescein (FL) is substituted for DmF as the donor moiety in the ET pair.

FIG. 38 provides growth curves for various PCR assays including the use of the 5' -nuclease probes shown in FIGS. 37A and 37B. The ordinate of the graph shown in fig. 38 represents the raw fluorescence, and the abscissa represents the cycle number. FIG. 39 is a graph of the growth curve shown in FIG. 38 normalized to the detected fluorescence. As shown, the trace corresponding to the reaction comprising the DmF probe and 100,000 initial copies of the template nucleic acid is labeled "DmF probe 100K", the trace corresponding to the reaction comprising the DmF probe and 1,000 initial copies of the template nucleic acid is labeled "DmF probe 1K", and the trace corresponding to the reaction comprising the DmF probe and 100 initial copies of the template nucleic acid is labeled "DmF probe 100". The trace corresponding to the reaction comprising the non-DmF probe and 100,000 initial copies of the template nucleic acid is labeled "non-DmF probe 100K", the trace corresponding to the reaction comprising the non-DmF probe and 1,000 initial copies of the template nucleic acid is labeled "non-DmF probe 1K", and the trace corresponding to the reaction comprising the non-DmF probe and 100 initial copies of the template nucleic acid is labeled "non-DmF probe 100". Negative controls in which the reaction mixture lacks template nucleic acid were also performed for both 5' -nuclease probes. The traces corresponding to these control reactions were labeled with "DmF probe and non-DmF probe controls". As shown in fig. 38 and 39, each PCR was performed in duplicate.

Example VII: additional hybridization Probe assays

This example shows performance characteristics of various combinations of donor and acceptor moieties in representative hybridization probe pairs. For example, FIG. 40 shows the donor and acceptor portions of these hybridization probe pairs. As shown, the donor probe 4000 and the acceptor probe 4002 hybridize to the single-stranded complementary sequence 4004. The donor probe used in this example included FAM, DmF or Dam HEX as the donor moiety, while the acceptor probe used in this example included LC-Red 610, JA-270, CY3.5, CY5 or CY5.5 as the acceptor moiety.

41A-D are graphs of emission scans obtained with the acceptor probes described herein (the ordinate of the graph represents absolute fluorescence, while the abscissa represents wavelength (nm)). Specifically, FIG. 41A is a graph of the emission scan obtained from the hybridization probe labeled with LC-Red 610, and FIG. 41B is a graph of the emission scan obtained from the hybridization probe labeled with JA-270. Furthermore, FIG. 41C is a graph of the emission scan obtained from the hybridization probe labeled CY5, and FIG. 41D is a graph of the emission scan obtained from the hybridization probe labeled CY 5.5.

FIG. 42A is a superimposed plot of emission scans obtained from hybridization probes labeled with FAM, one DmF molecule (6-carboxy isomer) (DmF), or two DmF molecules (6-carboxy isomer) (DmFx2) (ordinate represents absolute fluorescence and abscissa represents wavelength (nm)). The excitation wavelength (nm) of each trace is indicated (ex). FIG. 42B is an overlay plot of some of the emission scans shown in FIG. 42A, in which relative fluorescence emissions are provided.

Fig. 43A is a superimposed plot of excitation scans for hybridization probe assays involving an LC 610-labeled acceptor probe (maximum emission wavelength of 604nm) and a FAM, DmF or DmFx 2-labeled donor probe (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The excitation peaks for FAM, Dmf and LC-Red 610 are also shown. The emission wavelength (nm) of each trace is indicated (em). Also as shown, scans of negative controls including only acceptor probes (LC-Red 610 only, em ═ 604) in the reaction mixtures were also obtained. FIG. 43B is a superimposed plot of emission scans for these hybridization probe assays involving an acceptor probe labeled with LC-Red 610 and a donor probe labeled with FAM, DmF, or DmFx2 (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The excitation wavelength (nm) corresponding to each trace is indicated (ex). As also shown, scans of negative controls including only acceptor probes in the reaction mixture were also obtained at the excitation wavelengths of 495nm (LC-Red 610 only, ex ═ 495) and 515nm (LC-Red 610 only, ex ═ 515). FIG. 43C is an overlay plot of the emission scan shown in FIG. 43B, in which relative fluorescence emissions are provided.

Fig. 44A is a superimposed plot of excitation scans for hybridization probe assays involving acceptor probes labeled JA-270 (maximum emission wavelength 640nm) and donor probes labeled FAM, DmF or DmFx2 (ordinate represents absolute fluorescence and abscissa represents wavelength (nm)). The excitation peaks for FAM, Dmf and JA-270 are also shown. The emission wavelength (nm) of each trace is indicated (em). Also as shown, scans of negative controls including only acceptor probes (JA-270 only, em-640) in the reaction mixture were also obtained. FIG. 44B is a superimposed plot of emission scans for these hybridization probe assays involving acceptor probe labeled JA-270 and donor probe labeled FAM, DmF or DmFx2 (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The excitation wavelength (nm) corresponding to each trace is indicated (ex). Also as shown, scans of negative controls including only acceptor probes in the reaction mixture were also obtained at an excitation wavelength of 495nm (JA-270 only, ex 495) and an excitation wavelength of 515nm (JA-270 only, ex 515). FIG. 44C is an overlay plot of the emission scan shown in FIG. 44B, in which relative fluorescence emissions are provided.

Figure 45A is a superimposed plot of excitation scans for hybridization probe assays involving acceptor probes labeled with CY5 (maximum emission wavelength 663nm) and donor probes labeled with FAM, DmF or DmFx2 (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). Excitation peaks for FAM, DmF and CY5 are also shown. The emission wavelength (nm) of each trace is indicated (em). Also as shown, scans of negative controls including only the acceptor probe (CY 5 only, em 663) in the reaction mixture were also obtained. FIG. 45B is a superimposed plot of emission scans for these hybridization probe assays involving an acceptor probe labeled with CY5 and a donor probe labeled with FAM, DmF, or DmFx2 (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The excitation wavelength (nm) corresponding to each trace is indicated (ex). As also shown, scans of negative controls including only acceptor probes in the reaction mixture were also obtained at an excitation wavelength of 495nm (CY 5 only, ex 495) and an excitation wavelength of 515nm (CY 5 only, ex 515). FIG. 45C is an overlay plot of the emission scan shown in FIG. 45B, in which relative fluorescence emissions are provided.

Figure 46A is an overlay plot of excitation scans for hybridization probe assays involving acceptor probes labeled CY5.5 (maximum emission wavelength 702nm) and donor probes labeled FAM, DmF or DmFx2 (ordinate represents absolute fluorescence and abscissa represents wavelength (nm)). Excitation peaks for FAM, DmF and CY5.5 are also shown. The emission wavelength (nm) of each trace is indicated (em). Also as shown, scans of negative controls including only the acceptor probe (CY 5.5 only, em 702) in the reaction mixture were also obtained. FIG. 46B is a superimposed plot of emission scans for these hybridization probe assays involving an acceptor probe labeled CY5.5 and a donor probe labeled FAM, DmF or DmFx2 (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The excitation wavelength (nm) corresponding to each trace is indicated (ex). As also shown, scans of negative controls including only acceptor probes in the reaction mixture were also obtained at an excitation wavelength of 495nm (CY 5.5 only, ex 495) and an excitation wavelength of 515nm (CY 5.5 only, ex 515). FIG. 46C is an overlay plot of the emission scan shown in FIG. 46B, in which relative fluorescence emissions are provided.

Fig. 47A is a superimposed plot of excitation scans for hybridization probe assays involving an LC 610-labeled acceptor probe (maximum emission wavelength of 604nm) and a FAM, DmF or Dam HEX-labeled donor probe (i.e., 6-carboxy-amino pentachlorofluorescein) (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The emission wavelength (nm) of each trace is indicated (em). Also as shown, scans of negative controls including only acceptor probes (LC-Red 610 only, em 605) in the reaction mixture were also obtained. FIG. 47B is a superimposed plot of emission scans for these hybridization probe assays involving an acceptor probe labeled with LC-Red 610 and a donor probe labeled with FAM, DmF, or Dam HEX (the ordinate represents absolute fluorescence, and the abscissa represents wavelength (nm)). The excitation wavelength (nm) corresponding to each trace is indicated (ex). Also as shown, scans of negative controls including only donor probes in the reaction mixture were obtained as shown (FAM only, ex-495 nm; DmF only, ex-515 nm and Dam HEX only, ex-485 nm).

FIG. 48A is a superimposed plot of excitation scans for hybridization probe assays involving an acceptor probe labeled CY3.5 and a donor probe labeled FAM, DmF, or Dam HEX (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The emission wavelength (nm) of each trace is indicated (em). Also as shown, scans of negative controls including only the acceptor probe (CY 3.5 only, em 605) in the reaction mixture were also obtained. FIG. 48B is a superimposed plot of emission scans for these hybridization probe assays involving an acceptor probe labeled CY3.5 and a donor probe labeled FAM, DmF, or Dam HEX (the ordinate represents absolute fluorescence and the abscissa represents wavelength (nm)). The excitation wavelength (nm) corresponding to each trace is indicated (ex). Also as shown, scans of negative controls including only donor probes in the reaction mixture were obtained as shown (FAM only, ex-495 nm; DmF only, ex-515 nm and Dam HEX only, ex-485 nm).

Having thus described the invention in rather detail for the purposes of clarity and understanding, it will be apparent to those skilled in the art from a reading of this specification that various changes in form and detail can be made therein without departing from the true scope of the invention. For example, all of the above techniques and instruments may be used in various different combinations.

Claims (22)

1. A biomolecule comprising at least one oligonucleotide or at least one polynucleotide and at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorescein, or 5-carboxy-amino pentachlorofluorescein, the substantially non-fluorescent donor moiety is capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is sufficiently close thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy, wherein the peak visible absorptions of the substantially non-fluorescent donor moiety and the acceptor moiety differ by 100nm or more.

2. The biomolecule of claim 1, wherein the biomolecule comprises an acceptor moiety.

3. The biomolecule of claim 1 or 2, wherein the substantially non-fluorescent donor moiety and the acceptor moiety are not connected to each other by at least one linker moiety.

4. The biomolecule of any one of claims 1 to 3, wherein the substantially non-fluorescent donor moiety and/or acceptor moiety is linked to the biomolecule by at least one linker moiety.

5. The biomolecule of claim 4, wherein the oligonucleotide comprises a primer nucleic acid or a probe nucleic acid.

6. The biomolecule of claim 4, wherein the probe nucleic acid comprises a hybridization probe, a 5' -nuclease probe, or a hairpin probe.

7. A reaction mixture comprising at least one nucleotide, at least one primer nucleic acid and/or at least a first probe nucleic acid, wherein one or more of the nucleotides, primer nucleic acid or first probe nucleic acid comprises at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorescein, or 5-carboxy-amino pentachlorofluorescein, the substantially non-fluorescent donor moiety is capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is sufficiently close thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy.

8. The reaction mixture of claim 7, wherein the nucleotide, primer nucleic acid, or first probe nucleic acid comprises an acceptor moiety.

9. A reaction mixture comprising at least a first nucleic acid synthesis reagent comprising at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorescein, or 5-carboxy-amino pentachlorofluorescein, the substantially non-fluorescent donor moiety being capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy.

10. The reaction mixture of claim 9, wherein the first nucleic acid synthesis reagent comprises an acceptor moiety, the substantially non-fluorescent donor moiety and the acceptor moiety being connected to each other by at least one linker moiety.

11. A method of detecting a target nucleic acid, the method comprising:

(a) binding at least one probe nucleic acid to a target nucleic acid, wherein the probe nucleic acid comprises at least one substantially non-fluorescent donor moiety and at least one acceptor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorofluorofluorescein, or 5-carboxy-amino pentachlorofluorescein, and the acceptor moiety accepts non-fluorescent energy transferred from the substantially non-fluorescent donor moiety and emits light in response to the accepted non-fluorescent energy; and

(b) detecting light emitted from the acceptor moiety, thereby detecting the target nucleic acid.

12. The method of claim 11, wherein the method comprises amplifying at least a subsequence of the target nucleic acid prior to and/or during (b).

13. The method of claim 11, wherein the substantially non-fluorescent donor moiety and acceptor moiety are connected to each other by at least one linker moiety.

14. The method of claim 11, wherein the probe nucleic acid comprises a nucleic acid and a hybridization probe, a 5' -nuclease probe, or a hairpin probe.

15. A method of detecting a target nucleic acid, the method comprising:

(a) providing at least first and second probe nucleic acids, wherein the first probe nucleic acid comprises at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorofluorofluorescein, or 5-carboxy-amino pentachlorofluorescein, and wherein the second probe nucleic acid comprises at least one acceptor moiety;

(b) binding the first and second probe nucleic acids to the target nucleic acid such that the acceptor moiety accepts non-fluorescent energy transferred from the substantially non-fluorescent donor moiety and emits light in response to the accepted non-fluorescent energy; and

(c) detecting light emitted from the acceptor moiety, thereby detecting the target nucleic acid.

16. The method of claim 15, wherein the method comprises amplifying at least a subsequence of the target nucleic acid prior to and/or during (c).

17. The method of claim 15, wherein the first and/or second probe nucleic acid comprises a nucleic acid and a hybridization probe, a 5' -nuclease probe, or a hairpin probe.

18. A method of performing a proximity assay, the method comprising:

(a) providing at least first and second probe nucleic acids, wherein the first probe nucleic acid comprises at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorofluorofluorescein, or 5-carboxy-amino pentachlorofluorescein, and wherein the second probe nucleic acid comprises at least one acceptor moiety;

(b) placing the first and second probe nucleic acids in a first, relative position, wherein the first and second probe nucleic acids are sufficiently close to each other at the first position that the acceptor moiety accepts non-fluorescent energy transferred from the substantially non-fluorescent donor moiety and emits light in response to the accepted non-fluorescent energy;

(c) moving the first and second probe nucleic acids to at least a second, relative position; and

(d) the proximity assay is performed by monitoring light emitted from the receiving body portion before, after, and/or while the first and second probe nucleic acids are moved to the second position.

19. A method of extending a primer nucleic acid, the method comprising contacting a target nucleic acid with a primer nucleic acid

(a) At least one extendible nucleotide and/or at least one terminator nucleotide,

(b) at least one biocatalyst that catalyzes nucleotide incorporation; and

(c) at least one primer nucleic acid which is at least partially complementary to at least a subsequence of the target nucleic acid,

incubating under conditions in which a biocatalyst that catalyzes nucleotide incorporation extends a primer nucleic acid by incorporating an extendable nucleotide and/or a terminating nucleotide at a terminus of the extended primer nucleic acid to produce at least one extended primer nucleic acid, wherein the primer nucleic acid, extendable nucleotide and/or terminating nucleotide comprises at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino-pentachlorofluorofluorofluorofluorofluorescein, or 5-carboxy-amino-pentachlorofluorescein, the substantially non-fluorescent donor moiety being capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is in sufficient proximity thereto, such that the acceptor moiety emits light in response to the received non-fluorescent energy, thereby extending the primer nucleic acid.

20. The method of claim 19, wherein the primer nucleic acid, extendable nucleotide and/or terminator nucleotide comprises an acceptor moiety.

21. A kit comprising at least a first biomolecule comprising at least one oligonucleotide or at least one polynucleotide and at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorofluorofluorofluorescein, or 5-carboxy-amino pentachlorofluorescein, the substantially non-fluorescent donor moiety being capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is sufficiently proximal thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy.

22. An apparatus, the apparatus comprising:

(a) at least one container and/or solid support comprising at least one biomolecule, the biomolecule comprises at least one oligonucleotide or at least one polynucleotide and at least one substantially non-fluorescent donor moiety, wherein the substantially non-fluorescent donor moiety comprises one or more of 4 ', 5' -dimethoxy-6-carboxyfluorescein, 4 ', 5' -dimethoxy-5-carboxyfluorescein, 6-carboxy-amino pentachlorofluorescein, or 5-carboxy-amino pentachlorofluorescein, the substantially non-fluorescent donor moiety is capable of transferring non-fluorescent energy to at least one acceptor moiety when the acceptor moiety is sufficiently close thereto such that the acceptor moiety emits light in response to the accepted non-fluorescent energy;

(b) at least one radiation source positioned to direct electromagnetic radiation at the donor portion;

(c) at least one detection device positioned to detect light emitted from the receptor portion when the receptor portion is sufficiently proximate to the substantially non-fluorescent donor portion.