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WO2019204873A1 - Biocapteurs électroluminescents - Google Patents

Biocapteurs électroluminescents Download PDF

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Publication number
WO2019204873A1
WO2019204873A1 PCT/AU2019/050367 AU2019050367W WO2019204873A1 WO 2019204873 A1 WO2019204873 A1 WO 2019204873A1 AU 2019050367 W AU2019050367 W AU 2019050367W WO 2019204873 A1 WO2019204873 A1 WO 2019204873A1
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Prior art keywords
biosensor
molecular
binding
target molecule
amino acid
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English (en)
Inventor
Kirill Alexandrov
Zhong Guo
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University of Queensland UQ
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University of Queensland UQ
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Priority claimed from AU2018901359A external-priority patent/AU2018901359A0/en
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Publication of WO2019204873A1 publication Critical patent/WO2019204873A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • THIS INVENTION relates to biosensors. More particularly, this invention relates to light-emitting biosensors that are suitable for detection of one or more target molecules in a sample.
  • the biosensor molecule may also relate to the field of synthetic biology such as for constructing artificial cellular signalling networks.
  • Detection of target molecules or analytes in biological samples is central to diagnostic monitoring of health and disease. Key requirements of analyte detection are specificity and sensitivity, particularly when the target molecule or analyte is in a limiting amount or concentration in a biological sample.
  • specificity is provided by monoclonal antibodies which specifically bind the analyte.
  • Sensitivity is typically provided by a label bound to the specific antibody, or to a secondary antibody which assists detection of relatively low levels of analyte.
  • This type of diagnostic approach has become well known and widely used in the enzyme-linked immunosorbent sandwich assay (ELISA) format.
  • enzyme amplification can even further improve sensitivity such as by using a product of a proenzyme cleavage reaction catalyzing the same reaction.
  • Some examples of such “autocatalytic” enzymes are trypsinogen, pepsinogen, or the blood coagulation factor XII.
  • specificity antibodies are relatively expensive and can be difficult to produce with sufficient specificity for some analytes.
  • Polyclonal antibodies also suffer from the same shortcomings and are even more difficult to produce and purify on a large scale.
  • the present invention addresses a need to develop quantitative, relatively inexpensive and easily produced molecular biosensors that readily detect the presence and/or the activity of target molecules (e.g., analytes) on short time scales that are compatible with treatment regimes.
  • target molecules e.g., analytes
  • Such biosensors can either be applied singly or in multiplex to validate and/or diagnose molecular phenotypes with high specificity and great statistical confidence irrespective of the genetic background and natural variations in unrelated physiological processes.
  • Such molecular biosensors may be used in other testing procedures such as where the target molecule or analyte is an illicit drug or performance-enhancing substance and/or in screening assays.
  • Other applications of the biosensors may include the screening of molecules that promote or inhibit a binding interaction between proteins or between proteins and other biological molecules such as lipids, carbohydrates, metabolites, ions and nucleic acids.
  • the present invention provides a molecular biosensor that is particularly suited to incorporation into devices such as laboratory or point-of-care devices for analysis and transmission of diagnostic results.
  • One broad form of the invention relates to a molecular biosensor comprising a sensor and an enzyme that facilitates the emission of light upon binding a target molecule by the sensor.
  • the invention relates to a molecular biosensor comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.
  • the amino acid sequence of the enzyme is circularly permuted.
  • the circularly permuted enzyme comprises respective amino acid sequences connected by a linker amino acid sequence.
  • the linker amino acid sequence comprises a light-emitting molecule.
  • the light-emitting molecule is a dye molecule.
  • the light-emitting molecule is a fluorescent protein or fragment thereof.
  • light is emitted at a first wavelength in response to detecting, binding or interacting with a target molecule, which then triggers or activates emission of light at a second wavelength by the light-emitting molecule dye or fluorescent protein or fragment thereof.
  • the enzyme is a bioluminescent enzyme.
  • the enzyme is obtainable or derived from Oplophagus gracilirostris.
  • a preferred substrate is fumarazine.
  • a molecular biosensor comprising: a first molecular component comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more first sensors; and a second molecular component comprising one or more second sensors, whereby the first and second sensors can bind a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.
  • the first and second sensors can co-operatively bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.
  • the first molecular component and the second molecular component comprise a switch that facilitates activating enzyme catalytic activity following an interaction between the first molecular component and the second molecular component upon binding a target molecule.
  • the first molecular component comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand of the second molecular component such as a calmodulin-binding peptide or variant thereof, whereby binding of the peptide by calmodulin allosterically switches the enzyme into a more catalytic ally active state.
  • the enzyme is a bioluminescent enzyme.
  • the enzyme is obtainable or derived from Oplophagus gracilirostris.
  • a preferred substrate is fumarazine.
  • Another broad form of the invention relates to a biosensor comprising a sensor and a fluorescent protein that facilitates the emission of light upon binding a target molecule by the sensor.
  • this form relates to a molecular biosensor comprising at least one amino acid sequence of one or more fragments of a fluorescent protein and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.
  • the amino acid sequence of the fluorescent protein is circularly permuted, whereby the sensor interconnects the first and second fragments of the fluorescent protein.
  • the biosensor comprises first and second fragments of the fluorescent protein and a stabilizer that facilitates or stabilizes an interaction between the first and second fragments upon binding a target molecule.
  • the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3-binding peptide.
  • the molecular biosensor comprises a switch that facilitates an increase in fluorescence emission by the fluorescent protein.
  • the molecular biosensor comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide or variant thereof.
  • a ligand such as a calmodulin-binding peptide or variant thereof.
  • binding between calmodulin or variant and the ligand facilitates allosteric switching of the fluorescent protein to thereby increase emission of light by the fluorescent protein.
  • this form of the invention relates to a molecular biosensor comprising: a first molecular component comprising first and second fragments of a fluorescent protein and a first sensor; and a second molecular component comprising a second sensor, whereby the first and second sensors can bind a target molecule, and/or with each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.
  • the first and second sensors can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co operatively emitting light.
  • the first molecular component comprises a stabilizer that facilitates or stabilizes an interaction between the first and second fragments of the fluorescent protein.
  • the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3 -binding peptide.
  • the molecular biosensor may comprise another stabilizer, wherein the first molecular component comprises an amino acid sequence of calmodulin that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide of the second molecular component upon binding a target molecule.
  • a further aspect of the invention provides a molecular biosensor comprising: a first molecular component comprising a first fragment of a fluorescent protein and a first sensor; and a second molecular component comprising a first fragment of a fluorescent protein and a second sensor, whereby the first and second sensors can bind a target molecule, and/or with each other, to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.
  • the first and second sensors can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co operatively emitting light.
  • the molecular biosensor comprises a stabilizer that facilitates or stabilizes an interaction between the first and second molecular components of the biosensor.
  • the stabilizer comprises an amino acid sequence of calmodulin that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide of the second molecular component upon binding a target molecule.
  • a ligand such as a calmodulin-binding peptide of the second molecular component upon binding a target molecule.
  • a related aspect of the invention provides a method of producing an allosterically switchable protein that includes producing one or more chimeric proteins that comprise an amino acid sequence of at least a fragment of a protein of interest and an amino acid sequence of calmodulin, or a variant or fragment thereof.
  • This aspect also includes an allosterically switchable protein produced according to the method and/or a molecular biosensor comprising one or more of the allosterically switchable proteins.
  • a further aspect of the invention provides a biosensor device comprising one or more biosensors according to the aforementioned aspects immobilized or affixed to a support that is transparent to light emitted by the biosensor.
  • a yet further aspect of the invention provides a method of detecting a target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence of the target molecule in the sample.
  • Another yet further aspect of the invention provides a method of screening or identifying an inhibitory target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence an inhibitory target molecule that at least partly inhibits binding by the sensor(s).
  • the sensors may bind each other directly, which is inhibited by the inhibitory target molecule.
  • the inhibitory target molecule inhibits a binding interaction between the sensors and a target molecule.
  • inhibition is detected or measured as decrease in light emission.
  • a still yet further aspect of the invention provides a method of diagnosis of a disease or condition in an organism, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a biological sample obtained from the organism to thereby determine the presence or absence of a target molecule in the biological sample, determination of the presence or absence of the target molecule facilitating diagnosis of the disease or condition.
  • the organism may include plants and animals inclusive of fish, avians and mammals such as humans.
  • Another still yet further aspect of the invention provides a detection device that comprises a cell or chamber that comprises the biosensor or biosensor device of any of the aforementioned aspects.
  • a sample may be introduced into the cell or chamber to thereby facilitate detection of a target molecule.
  • the detection device is capable of providing an electrochemical, acoustic and/or optical signal that indicates the presence of the target molecule.
  • the detection device may further provide a disease diagnosis from a diagnostic target result by comprising:
  • the memory including computer readable program code components that, when executed by the processor
  • processor perform a set of functions including:
  • the detection device may further provide for communicating a diagnostic test result by comprising:
  • a related aspect of the invention provides an isolated nucleic acid encoding the biosensor of any of the aforementioned aspects..
  • Another related aspect of the invention provides a genetic construct comprising the isolated nucleic acid of the aforementioned aspect.
  • a further related aspect of the invention provides a host cell comprising the genetic construct of the aforementioned aspect.
  • indefinite articles ‘a’ and ‘ an’ are used here to refer to or encompass singular or plural elements or features and should not be taken as meaning or defining “one” or a“single” element or feature.
  • “about” refers to a tolerance or variation in a stated value or amount that does not appreciably or substantially affect function, activity or efficacy. Typically, the tolerance or variation is no more than 10%, 5%, 3%, 2%, or 1% above or below a stated value or amount.
  • FIG. 1 Introduction to NanoLuc.
  • the enzyme was isolated from a deep-water shrimp Oplophagus gracilirostris and has peak luminescence at around 460nm.
  • Bioluminescence from the NanoLuc (NLuc) system occurs when the optimized substrate called furimazine reacts with NLuc in the presence of molecular oxygen. This reaction yields furimamide and luminescence output.
  • FIG. 1 Development of an allosteric switch module based on Calmodulin (CaM)-NanoLuc chimera activated by calmodulin binding protein (CaM-BP).
  • the right panel represents titration of lOnM of CaM-NanoLuc chimeric protein with increasing concentrations of CaM-BP.
  • the data was fitted to a quadratic equation leading to a Kd of 17hM.
  • the panel below shows wells of a 96 well plate containing 100 qL of ImM CaM-NanoLuc and 10m1 furimazine solution (from Promega) in 20mM Tris-HCl pH 7.2, 20mM NaCl and lmM CaCl 2 .
  • the right well was supplemented with 5mM of CaM- BP.
  • FIG. 3 A two component biosensor based on CaM-NanoLuc allosterically switchable chimera.
  • A A schematic representation of the two component biosensor. The ligand (shown as a black star) induces dimerization of biosensor components and activation of NanoLuc activity.
  • B Titration of 200m1 10hM FKBP-CaM-NanoLuc biosensor mixed with 30nM FRB-CaM-BP component and 0.25m1 furimazine solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl and 0.5mM CaCF. The luminescence values were plotted against the concentration of the drug and fitted to a quadratic equation leading to a K d of lOnM.
  • Figure 4 Engineering of circular permutated NanoLuc and construction thereon based biosensors.
  • the bottom panel represents molecular representations of wt (left) and the circular permutated NanoLuc.
  • Figure 5. A single component rapamycin biosensor based on the circular permutated Nano Luc.
  • A Graphic representation of the biosensor where the NanoLuc component , FKBP and FRB are shown in ribbon representation.
  • FIG. 6 A single component alpha- amylase biosensor based on the circular permutated NanoLuc.
  • A Graphic representation of the biosensor where the NanoLuc component is shown in ribbon representation while VHH domains attached to the N and C terminus are shown as geometric shapes.
  • B Titration of 200m1 solution of lnM alpha- amylase biosensor supplemented with 0.25m1 furimazine stock solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl with the increasing concentrations of alpha- amylase. The data was fitted to a K d of 0.4nM.
  • FIG. 7 Comparison of the structures of FKBP:rapamycin:FRB complex with FKBP:tacrolimus:Calcineurin A/B complex. Due to the large size of the complex and its non-covalent nature the subunits of Calcineurin A/B were fused to form a single subunit entity that is more amenable to engineering.
  • FIG. 8 Structure and performance of Tacrolimus one component biosensor
  • A A model of a single component tacrolimus biosensor composed of circular permutated NanoLuc flanked with FKBP (displayed as ribbon) and a fusion of Calcineurin A and Calcineurin B proteins (displayed as a molecular surface).
  • B Titration of 200m1 solution of InM Tacroli us biosensor supplemented with 0.25ml furimazine stock solution in buffer containing 20mM Tris-HCl pH 7.2, 20mM NaCl with the increasing concentrations of the drug in the presence or absence of 50% serum. The data was fitted to a K d of 0.4nM.
  • C same as in (B) but in the presence or absence of 50% saliva.
  • Figure 9 Assessing the suitability of a single component tacrolimus biosensor for PoC applications.
  • A Assessing the time dependence of biosensor activation. The stock reaction containing InM Tacrolimus biosensor supplemented with 0.25m1/200m1 furimazine stock solution were mixed with the indicated concentrations of tacrolimus and incubated for indicated periods of time in the 96 well plate. The luminescence of the samples was then measured and the data was plotted against the concentration of Tacrolimus.
  • B The comparison of the luminescence yield of InM solution of the wild type recombinant NanoLuc, CaM-NanoLuc in the presence of lOOnM of CaM-BP and NanoLuc-based tacrolimus biosensor in the presence of 50nM of tacrolimus.
  • FIG. 10 Construction of NanoLuc biosensors with red-shifted emission
  • A The design sequence of converting the wild type NanoLuc into a BRET sensor. The circular permutation of NanoLuc followed by the insertion of a fluorescent protein domain (in this case EGFP) between de novo created N and C- terminus. The subsequent addition of the binding domains creates a biosensor that when activated by a ligand results in photon emission at 460nm that leads to fluorescent excitation of the fused EGFP that subsequently emits light with the emission maximum of 510nM.
  • B a fluorescent scan of InM solution of rapamycin biosensor constructed as shown in (A).
  • the red trace represents the emission of the biosensor in the absence of rapamycin while the green trace represents emission of the biosensor solution supplemented with 20nM of rapamycin.
  • the maximum emission of NanoLuc is indicated by an arrow.
  • C Titration of InM solution of rapamycin biosensor from A and B with increasing concentration of rapamycin. The luminescence (wavelength over 480nm) was recorded by applying filter BLUE1 (TECAN plate reader). And the determined values were plotted against the concentrations of rapamycin. The data was fitted to the quadratic equation leading to a Kd value of 0.5nM which is very close to the values obtained for the parental biosensor.
  • a biosensor based on the NanoLuc where a red fluorescent protein such as Cherry is inserted between the N and C -terminal parts of the circular permutated NanoLuc.
  • the protein serves as a linker and is used to convert the emission of the NanoLuc from blue (460nm) into the red emission (>600nm ).
  • B Same as in (A) but instead of red fluorescent protein a red organic dye is used as a luminescence acceptor and the fluorophore.
  • FIG. 12 Further approaches for extending the emission wavelength of the NanoLuc-based biosensors.
  • the bottom plot shows the emission scans of NanoLuc luminescence exposed to different compounds. The last two scans represent BRET from the compound F25 to the far-red small molecule organic dyes.
  • FIG 13 An alternative approach for measuring the luminescence of NanoLuc- based biosensors in the presence of lysed blood.
  • the biosensor is dried in a film on the transparent surface of a substrate such as a glass slide.
  • the drying matrix includes substances with high viscosity that restrict free diffusion.
  • the lysed blood sample containing the analyte and the NanoLuc substrate are added on top of the film rehydrating it and resulting in diffusion of the substrate and analyte to the biosensor. The emission was collected through the transparent side of the slide.
  • the calmodulin-calmodulin binding interaction acts as a stabilizer.
  • FIG. 15 Peptide“single component” biosensor based on cpGFP-CalM Ca.
  • the calmodulin-calmodulin binding peptide interaction acts as an allosteric switch.
  • FIG. 15 Rapamycin“two-component” biosensor based on the biosensor in FIG. 15. As in FIG. 15, the calmodulin-calmodulin binding peptide interaction acts as an allosteric switch.
  • Rapamycin“single component” biosensor based on FKBP-cpGFP-FRB.
  • FIG. 19 Generic approach for construction of protein switches.
  • the gene coding for a protein of interest (Pol) is used to create a library of calmodulin (CaM) insertion mutants.
  • CaM calmodulin
  • the present invention provides a biosensor which is capable of producing or generating light in response to detecting, binding or interacting with a target molecule.
  • the biosensor comprises an enzyme or enzyme fragment switchable between catalytically“inactive” and catalytically“active” states to thereby react with a substrate molecule to produce light.
  • the enzyme or enzyme fragment is a bioluminescent enzyme which has been engineered to enable switching between catalytically“inactive” and catalytically“active” states.
  • the enzyme is circularly permuted.
  • the circularly permuted enzyme comprises a linker amino acid sequence that may comprise a light- emitting molecule such as a dye or a fluorescent protein or fragment thereof.
  • a light- emitting molecule such as a dye or a fluorescent protein or fragment thereof.
  • light is emitted at a first wavelength in response to detecting, binding or interacting with a target molecule, which then triggers or activates emission of light at a second wavelength by the light-emitting molecule dye or fluorescent protein or fragment thereof.
  • the biosensor comprises respective fragments of a fluorescent protein, wherein binding of a target molecule causes the fragments to co operatively emit light.
  • the biosensor molecule(s) disclosed herein may have efficacy in molecular diagnostics wherein the“target molecule” is an analyte or other molecule of diagnostic value or importance.
  • the sensitivity of the biosensors disclosed herein may have particular efficacy in screening assays to detect target molecules that promote a binding interaction or those that inhibit a binding interaction.
  • Another application of the biosensor disclosed herein may be in synthetic biology applications for constructing multi-component artificial cellular signalling networks.
  • isolated material (such as a molecule) that has been removed from its natural state or otherwise been subjected to human manipulation.
  • Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
  • Isolated proteins and nucleic acids may be in native, chemical synthetic or recombinant form.
  • amino acid polymer By‘ protein” is meant an amino acid polymer.
  • the amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art.
  • A“ peptide” is a protein having less than fifty (50) amino acids.
  • A“ polypeptide” is a protein having fifty (50) or more amino acids.
  • A“fluorescent protein” as used herein may relate to any protein or fragment thereof that is capable of emitting light of a particular wavelength upon excitation by light of a different wavelength.
  • Fluorescent proteins may include those originally obtainable from animal and plant organisms such as of the genera Aequorea, Discosoma, Solanum, Montipora, Lobophyllia, Anemonia etc , which may include green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP) etc and also synthetically engineered variants that have desired spectral properties different to their naturally-occurring counterparts, such as mStrawberry, mOrange and mTomato, although without limitation thereto.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • BFP blue fluorescent protein
  • CFP cyan fluorescent protein
  • first and second are used in the context of respective, separate or discrete molecular components of the biosensor, such as sensors, enzymes, fluorescent protein fragments and/or binding moieties, it will be appreciated that these do not relate to any particular non-arbitrary ordering or designation that cannot be reversed. Accordingly, the structure and functional properties of the first component or second component disclosed herein could be those of a second component or a first component, respectively. Likewise, the structure and functional properties of a first sensor and a second sensor disclosed herein could be those of a second sensor and a first sensor, respectively. Similarly, the structure and functional properties of a first binding moiety and the second binding moiety disclosed herein could be those of a second binding moiety and a first binding moiety, respectively. It will also be appreciated that the biosensor may further comprise one or more other, non-stated molecular components.
  • a“ component” or“ molecular component’ is a discrete molecule that forms a separate part, portion or component of the biosensor.
  • each molecular component is, or comprises, a single, contiguous amino acid sequence (i.e a fusion protein or chimera).
  • a“ target molecule” may be any molecule detectable by the biosensor.
  • the target molecule may bind, or be bound or interact with the one or more sensors of the biosensor.
  • the target molecule may promote or enhance a binding interaction between the sensors.
  • the target molecule may at least partly prevent or inhibit a binding interaction between the sensors, or between the sensors and another target molecule, referred to herein as an“ inhibitory target molecule”.
  • a target molecule may be present in a“ sample”, which may be an isolate, specimen, extract, library or other mixture of compounds that potentially includes a target molecule, inclusive of inhibitory target molecules. These may include biological samples, dmg samples, molecular libraries of naturally-occurring molecules, synthetic libraries, combinatorial libraries and/or molecules produced from in silico libraries of molecular stmctures, although without limitation thereto.
  • One broad form of the invention relates to a molecular biosensor comprising a sensor and an enzyme that facilitates the emission of light upon binding a target molecule by the sensor.
  • the invention relates to a molecular biosensor comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more sensors that can bind or interact with a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.
  • an“ enzyme” is a protein having catalytic activity towards one or more substrate molecules.
  • the enzyme is capable of displaying catalytic activity towards a substrate molecule to thereby produce light.
  • the enzyme is a bioluminescent enzyme or one or more fragments thereof.
  • the enzyme is a luciferase, such as obtainable or derived from Oplophagus gracilirostris.
  • the luciferase is commercially available as NanoLuc ® .
  • a preferred substrate is fumarazine, which is converted to fumarazide by the luciferase, with emission of light at about 460nm. This is shown schematically in FIG.1.
  • catalytically active and“catalytically active state” may refer to absolute or relative amounts of enzyme activity that can be displayed or achieved by an enzyme or a fragment or portion thereof.
  • an enzyme is catalytically active or in a catalytically active state if it is capable of displaying specific enzyme activity towards a substrate molecule to produce light under appropriate reaction conditions.
  • catalytically inactive and“ catalytically inactive state” may refer to an enzyme, fragment or portion thereof that is substantially incapable of displaying specific enzyme activity towards a substrate molecule under appropriate reaction conditions.
  • the light produced would be substantially less compared to that produced by a corresponding catalytically active enzyme, or would be entirely absent.
  • the enzyme may switch from a“ catalytically active state” to a catalytically inactive state”.
  • a first particular aspect of the invention provides a molecular biosensor comprising at least one amino acid sequence of an enzyme capable of reacting with a substrate molecule, when in a catalytically active state to produce light; and one or more sensors that can bind or interact with a target molecule, and/or with each other, to thereby facilitate the enzyme reacting with the substrate molecule to produce light.
  • the enzyme is in a catalytically inactive state before the one or more sensor amino acid sequences bind or interact with a target molecule.
  • the enzyme is in a catalytically active state before a binding interaction between the one or more sensor amino acid sequences is inhibited by an inhibitory target molecule.
  • said biosensor is in the form a single, contiguous amino acid sequence, such as in the form of a fusion protein or chimeric protein.
  • the enzyme is a bioluminescent enzyme or one or more fragments thereof.
  • the enzyme is a luciferase, such as obtainable or derived from Oplophagus gracilirostris.
  • the luciferase is commercially available as NanoLuc ® .
  • a preferred substrate is fumarazine, which is converted to fumarazide by the luciferase, with emission of light at about 460nm.
  • the amino acid sequence of the enzyme is circularly permuted.
  • the circularly permuted amino acid sequence comprises an amino acid sequence that is normally at or near the C terminus of the enzyme located N-terminal of an amino acid sequence that is normally at or near the N terminus of the enzyme.
  • the circularly permuted amino acid sequence comprises: (i) an amino acid sequence that is normally at or near the C terminus of the enzyme located N-terminal of an amino acid sequence that is normally at or near the N terminus of the enzyme; and (ii) a linker or spacer amino acid sequence between said amino acid sequence that is normally at or near the C terminus of the enzyme and said amino acid sequence that is normally at or near the N terminus of the enzyme.
  • a general embodiment provides a circularly permuted enzyme according to the following general formula:
  • X-C is normally the C-terminal amino acid sequence of the enzyme, or fragment thereof;
  • N-Y is normally the N-terminal amino acid sequence of the enzyme, or fragment thereof.
  • the linker is an amino acid sequence contiguous with X-C and N-Y.
  • FIGS. 4-6, 10 and 11 are schematically shown in FIGS. 4-6, 10 and 11.
  • Circular permutation disrupts enzyme activity, which activity is then rescued by re-association of the enzyme portion, mediated by the sensor(s) detecting or binding a target molecule.
  • a proposed molecular mechanism is that the b-strand fused to the binding moiety can be pulled out of the protein due to the solvation forces and binding of the target molecule (in this case a amylase) which leads to the forced re association of the enzyme portions leading to reconstitution of the enzyme and hence activation of the catalytic activity of the enzyme.
  • the target molecule in this case a amylase
  • spectral filtering can occur whereby molecules in a biological sample can absorb light emitted by the biosensor.
  • the issue of spectral filtering by haemoglobin is a common problem.
  • an embodiment of the present invention utilizes light emitted at a first wavelength by the enzyme reacting with the substrate molecule to activate emission of light at a second wavelength by a light-emitting molecule.
  • This phenomenon is termed Bioluminescence Resonance Energy Transfer or BRET.
  • the light-emitting molecule may be referred to as a“wavelength converter”.
  • the linker amino acid sequence comprises the light-emitting molecule.
  • the light-emitting molecule is a fluorescent protein or fragment thereof, such as those hereinbefore described.
  • Non-limiting examples include green fluorescent protein (GFP) and red fluorescent protein (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the biosensor of this embodiment shifts the emission wavelength into the red and far-red spectrum in order to reduce haemoglobin-mediated signal filtering. It will therefore be appreciated that this principle may be applied to any light-emitting biosensor where the emitted light is of a wavelength that may be absorbed or attenuated by molecules potentially present in a biological sample.
  • the light-emitting molecule is a dye molecule.
  • the dye molecule may be an organic dye molecule.
  • a small organic red dye may be conjugated to the enzyme using site selective chemistry.
  • site selective chemistry A non-limiting example is shown in FIG. 11. This may be achieved using thiol chemistry in case cysteines are absent from the sequence of the protein biosensor (or present at positions where they can be mutated to serine without compromising the structure or function of the protein).
  • codon reassignment can be used to incorporate a biorthogonal moiety, such as 4-Azidophenylalanine, that can be used for dye conjugation via click chemistry.
  • An alternative embodiment would include furimazine derivatives with red shifted emission maximum, as recently reported by Shakhmin l al, 2017, Org. Biomol. Chem. 15 8559.
  • Such substrates can be used for forming BRET pairs resulting in far-red shifted emission as schematically shown in FIG.12.
  • Another form of the invention relates to a biosensor comprising: a first molecular component comprising at least one amino acid sequence of an enzyme that is capable of reacting with a substrate molecule to produce light and one or more first sensor amino acid sequences; and a second molecular component comprising one or more second sensor amino acid sequences, whereby the first and second sensor amino acid sequences can bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light.
  • the first and second sensor amino acid sequences can co-operatively bind a target molecule to thereby facilitate the enzyme reacting with the substrate molecule to produce light. Accordingly, the binding interaction between the target molecule and the binding moieties of the sensor amino acid sequences facilitates co localization of the first and second molecular components.
  • the first molecular component comprises an amino acid sequence of calmodulin that is capable of binding or interacting with a ligand of the second molecular component such as a calmodulin-binding peptide.
  • the first molecular component and the second molecular component comprise a switch that facilitates activating enzyme catalytic activity following an interaction between the first molecular component and the second molecular component upon binding a target molecule.
  • the first molecular component comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand of the second molecular component such as a calmodulin-binding peptide or variant thereof, whereby binding of the ligand by calmodulin allosterically switches the enzyme into a more catalytic ally active state.
  • FIG. 3 A non-limiting example of a “two-component” biosensor comprising a bioluminescent enzyme“switched” by this calmodulin- calmodulin peptide interaction is shown in FIG. 3.
  • Another broad form of the invention relates to a biosensor comprising a sensor and a fluorescent protein that facilitates the emission of light upon binding a target molecule by the sensor.
  • this form relates to a biosensor comprising at least one amino acid sequence of one or more respective fragments of a fluorescent protein and one or more sensors that can bind or interact with a target molecule to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.
  • the biosensor may further comprise one or more stabilizers.
  • the stabilizer may be, or comprise, any molecules that can bind or interact, such as“ complementary binding partners”. Preferably, this binding or interaction is non- covalent and reversible. These may include molecules that engage in ligand-receptor binding, calmodulin binding partners, protein-protein interaction domains (e.g SH3 domains and their proline-rich binding domains, leucine zippers and other dimerization domains, PDZ domains, LIM domains and their binding peptides) and antigen- antibody binding partners and affinity clamps although without limitation thereto.
  • the biosensor comprises first and second fragments of the fluorescent protein. In some embodiments, the sensor interconnects or is disposed between the first and second fragments of the fluorescent protein. In one embodiment, the biosensor comprises first and second fragments of the fluorescent protein and a stabilizer that facilitates or stabilizes an interaction between the first and second fragments.
  • the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3 -binding peptide.
  • FIG. 15 A non-limiting example is shown in FIG. 15.
  • the biosensor may further comprise a switch that facilitates activating fluorescence emission by the fluorescent protein following binding a target molecule.
  • the molecular biosensor comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand such as a calmodulin-binding peptide or variant thereof, whereby binding of the ligand by calmodulin allosterically activates fluorescence emission by the fluorescent protein.
  • the amino acid sequence of calmodulin or a variant thereof interconnects respective fragments of the fluorescent protein.
  • FIG. 15 A non-limiting example is shown in FIG. 15.
  • the fluorescent protein is circularly permuted. Circular permutation disrupts fluorescence emission activity, which activity is then rescued by re-association of the fluorescent protein portions, mediated by the sensor(s) detecting or binding a target molecule.
  • FIG. 17 A non-limiting example is shown in FIG. 17.
  • this form of the invention relates to a molecular biosensor comprising: a first molecular component comprising first and second fragments of a fluorescent protein and one or more first sensors; and a second molecular component comprising one or more second sensors, whereby the first and second sensors can bind a target molecule to thereby facilitate the respective fragments of the fluorescent protein co-operatively emitting light.
  • the first and second sensors can co-operatively bind a target molecule to thereby facilitate the respective fluorescent protein fragments co operatively emitting light.
  • the first and second fragments of the fluorescent protein molecular comprise a stabilizer that facilitates or stabilizes an interaction between the first and second fragments of the fluorescent protein upon binding a target molecule.
  • stabilizers and “complementary binding partners” as hereinbefore described.
  • the stabilizer comprises an amino acid sequence of an SH3 domain that is capable of non-covalently and reversibly binding or interacting with a peptide of the second molecular component, such as an SH3 -binding peptide.
  • a peptide of the second molecular component such as an SH3 -binding peptide.
  • FIG. 16 A non- limiting example is shown in FIG. 16.
  • the biosensor may further comprise a switch that facilitates activating fluorescence emission by the fluorescent protein following binding a target molecule.
  • the molecular biosensor comprises an amino acid sequence of calmodulin or a variant thereof that is capable of non-covalently and reversibly binding or interacting with a ligand, such as a calmodulin-binding peptide or variant thereof, whereby binding of the ligand by calmodulin allosterically activates fluorescence emission by the fluorescent protein.
  • the amino acid sequence of calmodulin or a variant thereof interconnects respective fragments of the fluorescent protein.
  • FIG. 15 A non-limiting example is shown in FIG. 15.
  • this form of the invention relates to a molecular biosensor comprising: a first molecular component comprising at least one amino acid sequence of a first fragment of a fluorescent protein and one or more first sensors; and a second molecular component comprising at least one amino acid sequence of a second fragment of a fluorescent protein and one or more second sensors, whereby the first and second sensors can bind a target molecule to thereby facilitate the respective first and second fragments of the fluorescent protein co-operatively emitting light.
  • the first and second sensor amino acid sequences can co-operatively bind a target molecule to thereby facilitate the first and second fluorescent protein fragments co-operatively emitting light.
  • the molecular biosensor comprises one or more stabilizers that facilitate or stabilize an interaction between the first and second components upon binding a target molecule.
  • stabilizers that facilitate or stabilize an interaction between the first and second components upon binding a target molecule.
  • the stabilizer comprises a calmodulin amino acid sequence which is capable of reversible and releasably binding a ligand such as a calmodulin-binding peptide.
  • the first molecular component comprises the calmodulin amino acid sequence and the second molecular component comprises the ligand, such as a calmodulin-binding peptide amino acid sequence.
  • FIG. 14 A non-limiting example is shown in FIG. 14.
  • first and second fragments of a“fluorescent protein” could be of the same or different fluorescent protein.
  • molecular biosensors comprise: (i) an enzyme that can react with a substrate to produce light: or (ii) respective fragments of a fluorescent protein; and comprise one or more sensors that facilitate detection of a target molecule.
  • the one or more sensors can bind or interact with a target molecule comprise one or more binding moieties that can bind or interact with the target molecule, or which can directly interact or bind in the absence of a target molecule.
  • the one or more binding moieties can co-operatively bind or interact with the target molecule.
  • binding moiety or“ binding moieties” refer to one or a plurality of molecules or biological or chemical components or entities that are capable of recognizing and/or binding each other, or one or more target molecules.
  • Binding moieties may be proteins, nucleic acids (e.g single-stranded or double- stranded DNA or RNA), sugars, oligosaccharides, polysaccharides or other carbohydrates, lipids or any combinations of these such as glycoproteins, PNA constructs etc or molecular components thereof
  • the binding moieties comprise an amino acid sequence of at least a fragment of any protein or protein fragment or domain that can bind or interact directly, or bind to a target molecule.
  • the binding moiety may be, or comprise a protein such as a peptide, antibody, antibody fragment or any other protein scaffold that can be suitably engineered to create or comprise a binding portion, domain or region (e.g. reviewed in Binz et al., 2005 Nature Biotechnology, 23, 1257-68.) which binds a target molecule.
  • binding moieties may be, or comprise: (i) an amino acid sequence of a ligand binding domain of a receptor responsive to binding of a target molecule such as a cognate growth factor, cytokine, a hormone (e.g.
  • an amino acid sequence of an ion or metabolite transporter capable of, or responsive to, binding of a target molecule such as an ion or metabolite (e.g a Ca 2+ -binding protein such as calmodulin or calcineurin or a glucose transporter);
  • a zinc finger amino acid sequence responsive to zinc-dependent binding a DNA target molecule e.g a Ca 2+ -binding protein such as calmodulin or calcineurin or a glucose transporter
  • a zinc finger amino acid sequence responsive to zinc-dependent binding a DNA target molecule e.g a Ca 2+ -binding protein such as calmodulin or calcineurin or a glucose transporter
  • a zinc finger amino acid sequence responsive to zinc-dependent binding a DNA target molecule e.g a Ca 2+ -binding protein such as calmodulin or calcineurin or a glucose transporter
  • a zinc finger amino acid sequence responsive to zinc-dependent binding a DNA target molecule
  • the binding moieties comprise one or a plurality epitopes that can be bind or be bound by an antibody target molecule.
  • the binding moieties may be or comprise an antibody or antibody fragment, inclusive of monoclonal and polyclonal antibodies, recombinant antibodies, Fab and Fab’ 2 fragments, diabodies and single chain antibody fragments (e.g. scVs), although without limitation thereto.
  • the first and second binding moieties may be or comprise respective antibodies or antibody fragments that bind a target molecule.
  • the binding moieties may be or comprise an antibody-binding molecule, wherein the antibody(ies) has specificity for a target molecule.
  • the antibody-binding molecule preferably comprises an amino acid sequence of protein A, or a fragment thereof (e.g a ZZ domain), which binds an Fc portion of the antibody.
  • the respective binding moieties are capable of binding, interacting or forming a complex with the same target molecule.
  • the “same” target molecule can have respective, different moieties, subunits, domains, ligands or epitopes that can be bound by the respective binding moieties to thereby co localize and activate enzyme activity.
  • the target molecule may be any ligand, analyte, small organic molecule, ion, epitope, domain, fragment, subunit, moiety or combination thereof, such as a protein inclusive of antibodies and antibody fragments, antigens, enzymes, phosphoproteins, glycoproteins, lipoproteins and glycoproteins, lipid, phospholipids, carbohydrates inclusive of simple sugars, disaccharides and polysaccharides, nucleic acids, nucleoprotein or any other molecule or analyte.
  • drugs and other pharmaceuticals including antibiotics, banned substances, illicit drugs or drugs of addiction, chemotherapeutic agents and lead compounds in drug design and screening, molecules and analytes typically found in biological samples such as biomarkers, tumour and other antigens, receptors, DNA-binding proteins inclusive of transcription factors, hormones, neurotransmitters, growth factors, cytokines, receptors, metabolic enzymes, signaling molecules, nucleic acids such as DNA and RNA, membrane lipids and other cellular components, pathogen-derived molecules inclusive of viral, bacterial, protozoan, fungal and worm proteins, lipids, carbohydrates and nucleic acids, although without limitation thereto.
  • the“same” target molecule can be bound by different, respective binding moieties.
  • the target molecule may be an inhibitory target molecule that at least partly prevents or inhibits a binding interaction between the sensor binding moieties.
  • a first binding moiety is an FKBP amino acid sequence and a second binding moiety comprises a rapamycin-binding FRB amino acid sequence.
  • the biosensor of this embodiment is capable of detecting, binding or interacting with rapamycin.
  • a first binding moiety comprises a rapamycin- binding FKBP amino acid sequence and a second binding moiety comprises one or more calcineurin amino acid sequences.
  • the one or more calcineurin amino acid sequences comprise a calcineurin A and a calcineurin B amino acid sequence preferably comprising an intervening linker amino acid sequence.
  • the biosensor of this embodiment is capable of detecting, binding or interacting with tacrolimus (FK-506).
  • the target molecule is an enzyme such as a amylase.
  • the first and second binding moieties are, respectively the camelid antibodies VHH1 and VHH2.
  • the target molecule is a secretory protein such as Human Serum Albumin.
  • the first and second binding moieties are, respectively the camelid antibodies VHH1 and bacterial albumin binding module.
  • the target molecule is a small organic molecule such as rapamycin.
  • the first and second binding moieties are, respectively the FKBP and FRB.
  • the target molecule is a small organic molecule such as FK506 or tacrolimus.
  • the first and second binding moieties are, respectively, the FKBP and a Calcineurin A/B complex.
  • the target molecule is a small organic molecule such as cyclosporin.
  • the first and second binding moieties are, respectively, a peptidyl prolyl cis trans isomerase A and Calcineurin A/B complex.
  • the target molecule is a small organic molecule such as Vitamin D.
  • the first and second binding moieties are, respectively, a Vitamin D binding protein that undergoes conformational transition when Vitamin D binds to it while the second moiety represents a protein domain that binds with much higher affinity to the Vitamin D binding protein: Vitamin D complex than to the apo form of the Vitamin D binding protein.
  • the binding moieties may be selected to identify previously unknown molecules that can bind or interact with the binding moieties.
  • the biosensor may facilitate screening for one or more molecules that enhance or enable a binding interaction between the binding moieties.
  • biosensors comprising FKBP amino acid sequence and FRB amino acid sequences may be used to identify new immunosuppressive molecules from a library of compounds.
  • the binding moieties may be capable of directly binding or interacting without binding a target molecule.
  • the biosensor may facilitate screening for one or more inhibitors that prevent binding between the binding moieties.
  • biosensors and the molecular components thereof described herein may be, or comprise, contiguous amino acid sequences such as in the form of chimeric proteins or fusion proteins as are well understood in the art.
  • respective amino acid sequences e.g binding moieties, enzyme amino acid sequences, protease amino acid sequences etc
  • respective amino acid sequences may be discrete or separate amino acid sequences linked or connected by spacers or linkers (e.g. amino acids, amino acid sequences, nucleotides, nucleotide sequences or other molecules) to optimize features or activities such as target molecule recognition, binding and enzyme activity or inhibition, although without limitation thereto.
  • Non-limiting examples of amino acid sequences inclusive of enzyme amino acid sequences, engineered mutants, linkers, protease cleavage sites, and binding moieties are provided in SEQ ID NOS: l-14.
  • biosensor molecules that are variants of the embodiments described herein, or which comprise variants of the constituent enzyme, fluorescent protein, sensor and/or other protein and peptide (e.g calmodulin, calmodulin-binding peptide) amino acid sequences disclosed herein.
  • such variants have at least 80%, at least 85%, preferably at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% sequence identity with any of the amino acid sequences disclosed herein, such as SEQ ID NOS: l-14 or portions thereof.
  • conservative amino acid variations may be made without an appreciable or substantial change in function.
  • conservative amino acid substitutions may be tolerated where charge, hydrophilicity, hydrophobicity, side chain “bulk”, secondary and/or tertiary structure (e.g. helicity), target molecule binding, enzyme or fluorescence activity are substantially unaltered or are altered to a degree that does not appreciably or substantially compromise the function of the biosensor.
  • Variants of the invention are selected to be functional and so retain or substantially retain catalytic activity, or the ability to reconstitute such catalytic activity when provided together with suitable further components of a biosensor as described above.
  • Variants of the non-covalently associating amino acid sequences (such as first and second fragment sequences) described herein are selected to retain the ability to reconstitute a stable enzyme or fluorescent protein when provided in combination with their respective binding partner sequence.
  • sequence identity is used herein in its broadest sense to include the number of exact amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
  • Sequence identity may be determined using computer algorithms such as GAP, BESTFIT, FASTA and the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995- 1999).
  • Protein fragments may comprise up to 5%, 10%, 15%, 20%, 25%, 30%, 35%,
  • the protein fragment may comprise up to 5, 10, 20, 40, 50, 70, 80, 90, 100, 120, 150, 180 200, 220, 230. 250, 280, 300, 330, 350, 400 or 450 amino acids of an amino acid sequence disclosed herein, such as SEQ ID NOS: l-14.
  • a further aspect of the invention provides a biosensor device comprising one or more biosensors according to the aforementioned aspects immobilized or affixed to a support that is substantially transparent to light emitted by the biosensor.
  • the support may be or include a slide, chip, wafer, strip, plate or other structure which is formed of a material that is substantially transparent to light emitted by the biosensor.
  • FIG. 13 An example is schematically shown in FIG. 13.
  • the biosensor is dried as a film on a substantially transparent surface of the support.
  • the biosensor molecule may be applied in a“drying matrix” that includes substances with high viscosity that restricts free diffusion.
  • a sample containing a target molecule and an enzyme substrate are added on top of the film, thereby rehydrating it and resulting in diffusion of the substrate and target molecule to the biosensor.
  • the emitted light is collected via the substantially transparent side of the device.
  • a further aspect of the invention provides a kit or composition comprising one or more biosensors disclosed herein, optionally in combination with one or more substrate molecules.
  • the kit is for detecting a target molecule is a sample to thereby determine the presence or absence of the target molecule in the sample.
  • the invention provides a method of detecting a target molecule, said method including the step of contacting the composition of the aforementioned aspect with a sample to thereby determine the presence or absence of the target molecule in the sample.
  • the sample is a biological sample.
  • Biological samples may include organ samples, tissue samples, cellular samples, fluid samples or any other sample obtainable, obtained, derivable or derived from an organism or a component of the organism.
  • the biological sample can comprise a fermentation medium, feedstock or food product such as for example, but not limited to, dairy products.
  • the biological sample is obtainable from a mammal, preferably a human.
  • the biological sample may be a fluid sample such as blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid or am ni otic fluid, a tissue sample such as a tissue or organ biopsy or may be a cellular sample such as a sample comprising red blood cells, lymphocytes, tumour cells or skin cells, although without limitation thereto.
  • a particular type of biological sample is a pathology sample.
  • the enzyme activity of the biosensor is not substantially inhibited by components of the sample (e.g. serum proteins, metabolites, cells, cellular debris and components, naturally-occurring protease inhibitors etc).
  • components of the sample e.g. serum proteins, metabolites, cells, cellular debris and components, naturally-occurring protease inhibitors etc.
  • the biosensor and/or methods of use may be applicable to drug testing such as for detecting the use of illicit drugs of addiction (e.g cannabinoids, amphetamines, cocaine, heroin etc.) and/or for the detection of performance-enhancing substances in sport and/or masking agents that are typically used to avoid detection of performance-enhancing substances.
  • drugs of addiction e.g cannabinoids, amphetamines, cocaine, heroin etc.
  • performance-enhancing substances in sport and/or masking agents that are typically used to avoid detection of performance-enhancing substances.
  • This may be applicable to the detection of banned performance -enhancing substances in humans and/or other mammals such as racehorses and greyhounds that may be subjected to illicit“doping” to enhance performance.
  • the target molecule promotes an interaction between targeting moieties.
  • the sample may comprise a library of compounds that may comprise at least one target molecule that increases, enhances or promotes a binding interaction between the binding moieties.
  • “immunosuppressant” biosensors could be used to identify alternative chemical structures that facilitate binding between the binding moieties.
  • a further aspect provides a method of screening or identifying an inhibitory target molecule, said method including the step of contacting the biosensor or biosensor device of any of the aforementioned aspects with a sample to thereby determine the presence or absence an inhibitory target molecule that at least partly inhibits the sensor(s).
  • the method may be a screening assay to identify molecules in a library are potential inhibitors of binding between the binding moieties, in which case the biosensor may be used to measure or detect binding inhibition.
  • light emission would decrease in the presence of the inhibitory target molecule.
  • the biosensor and/or methods of use are for diagnosis of a disease or condition of a mammal, such as a human.
  • a preferred aspect of the invention provides a method of diagnosis of a disease or condition in a human, said method including the step of contacting the composition of the aforementioned aspect with a biological sample obtained from the human to thereby determine the presence or absence of a target molecule in the biological sample, wherein determination of the presence or absence of the target molecule facilitates diagnosis of the disease or condition.
  • the disease or condition may be anywhere detection of a target molecule assists diagnosis.
  • target molecules or analytes include blood coagulation factors such as previously described, kallikreins inclusive of PSA, matrix metalloproteinases, viral and bacterial proteases, antibodies, glucose, triglycerides, lipoproteins, cholesterol, tumour antigens, lymphocyte antigens, autoantigens and autoantibodies, drugs, salts, creatinine, blood serum or plasma proteins, pesticides, uric acid, products and intermediates of human and animal metabolism and metals.
  • blood coagulation factors such as previously described, kallikreins inclusive of PSA, matrix metalloproteinases, viral and bacterial proteases, antibodies, glucose, triglycerides, lipoproteins, cholesterol, tumour antigens, lymphocyte antigens, autoantigens and autoantibodies, drugs, salts, creatinine, blood serum or plasma proteins, pesticides, uric acid, products and intermediates of human and animal metabolism and metals
  • This preferred aspect of the invention may be adapted to be performed as a “point of care” method whereby determination of the presence or absence of the target molecule may occur at a patient location which is then either analysed at that location or transmitted to a remote location for diagnosis of the disease or condition.
  • a still yet further aspect of the invention provides a detection device that comprises a cell or chamber that comprises the biosensor of any of the aforementioned aspects.
  • a sample may be introduced into the cell or chamber to thereby facilitate detection of a target molecule.
  • the detection device is capable of providing an electrochemical, acoustic and/or optical signal that indicates the presence of the target molecule.
  • the detection device may further provide a disease diagnosis from a diagnostic target result by comprising:
  • the memory including computer readable program code components that, when executed by the processor
  • processor perform a set of functions including:
  • the detection device may further provide for communicating a diagnostic test result by comprising:
  • Diagnostic aspects of the invention may also be in the form of a kit comprising one or a plurality of different biosensors capable of detecting one or a plurality of different target molecules.
  • a kit may comprise an array of different biosensors capable of detecting a plurality of different target molecules.
  • the kit may further comprise one or more amplifier molecules, deactivating molecules and/or labeled substrates, as hereinbefore described.
  • the kit may also comprise additional components including reagents such as buffers and diluents, reaction vessels and instructions for use.
  • a further aspect of the invention provides an isolated nucleic acid which encodes an amino acid sequence of the biosensor of the invention, or a variant thereof as hereinbefore defined.
  • nucleic acid designates single-or double-stranded mRNA, RNA, cRNA, RNAi, siRNA and DNA inclusive of cDNA, mitochondrial DNA (mtDNA) and genomic DNA.
  • A“ polynucleotide” is a nucleic acid having eighty (80) or more contiguous nucleotides, while an “ oligonucleotide” has less than eighty (80) contiguous nucleotides.
  • A“primer” is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid“template” and being extended in a template-dependent fashion by the action of a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
  • a “probe” may be a single or double-stranded oligonucleotide or polynucleotide, suitably labelled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.
  • the invention also provides variants and/or fragments of the isolated nucleic acids.
  • Variants may comprise a nucleotide sequence at least 70%, at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with any nucleotide sequence disclosed herein.
  • nucleic acid variants may hybridize with the nucleotide sequence of with any nucleotide sequence disclosed herein, under high stringency conditions.
  • Fragments may comprise or consist of up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95-99% of the contiguous nucleotides present in any nucleotide sequence disclosed herein.
  • Fragments may comprise or consist of up to 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 950, 1000, 1050, 1100, 1150, 1200, 1350 or 1300 contiguous nucleotides present in any nucleotide sequence disclosed herein.
  • the invention also provides“ genetic constructs” that comprise one or more isolated nucleic acids, variants or fragments thereof as disclosed herein operably linked to one or more additional nucleotide sequences.
  • a “genetic construct” is an artificially created nucleic acid that incorporates, and/or facilitates use of, an isolated nucleic acid disclosed herein.
  • such constructs may be useful for recombinant manipulation, propagation, amplification, homologous recombination and/or expression of said isolated nucleic acid.
  • a genetic construct used for recombinant protein expression is referred to as an " expression construct", wherein the isolated nucleic acid to be expressed is operably linked or operably connected to one or more additional nucleotide sequences in an expression vector.
  • An “expression vector” may be either a self-replicating extra-chromosomal vector such as a plasmid, or a vector that integrates into a host genome.
  • the one or more additional nucleotide sequences are regulatory nucleotide sequences.
  • operably linked or “operably connected” is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the nucleic acid to be expressed to initiate, regulate or otherwise control expression of the nucleic acid.
  • Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
  • One or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences and enhancer or activator sequences.
  • Constitutive or inducible promoters as known in the art may be used and include, for example, nisin-inducible, tetracycline-repressible, IPTG-inducible, alcohol- inducible, acid-inducible and/or metal-inducible promoters.
  • the expression vector comprises a selectable marker gene.
  • Selectable markers are useful whether for the purposes of selection of transformed bacteria (such as bla, kanR, ermB and tetR ) or transformed mammalian cells (such as hygromycin, G418 and puromycin resistance).
  • Suitable host cells for expression may be prokaryotic or eukaryotic, such as bacterial cells inclusive of Escherichia coli (DH5a for example), yeast cells such as S. cerivisiae or Pichia pastoris, insect cells such as SF9 cells utilized with a baculovirus expression system, or any of various mammalian or other animal host cells such as CHO, BHK or 293 cells, although without limitation thereto.
  • prokaryotic or eukaryotic such as bacterial cells inclusive of Escherichia coli (DH5a for example), yeast cells such as S. cerivisiae or Pichia pastoris, insect cells such as SF9 cells utilized with a baculovirus expression system, or any of various mammalian or other animal host cells such as CHO, BHK or 293 cells, although without limitation thereto.
  • telomeres e.g. lipofectin, lipofectamine
  • protoplast fusion e.g. lipofectin, lipofectamine
  • microinjection or microparticle bombardment e.g. electroporation, heat shock, calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-based transfection (e.g. lipofectin, lipofectamine), protoplast fusion, microinjection or microparticle bombardment, as are well known in the art.
  • Purification of the recombinant biosensor molecule may be performed by any method known in the art.
  • the recombinant biosensor molecule comprises a fusion partner (preferably a C-terminal His tag) which allows purification by virtue of an appropriate affinity matrix, which in the case of a His tag would be a nickel matrix or resin.
  • bioluminescent proteins The biomedical field has greatly benefited from the discovery of bioluminescent proteins.
  • bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence based molecular imaging.
  • these systems are based on Firefly and Renilla lucif erases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency.
  • NanoLuc a novel bioluminescent enzyme, offers several advantages over established systems, including enhanced stability, smaller size, and > 150-fold increase in luminescence 1 .
  • the substrate for NanoLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging and target molecule detection.
  • NanoLuc The very high quantum yield of NanoLuc makes it an attractive platform for biosensor development. Based on our previous experience we first decided to test if NanoLuc could be converted into the allosteric switch module. To this end we analyzed the high resolution structure of NanoLuc and inserted a calmodulin (CaM) domain between critical structural elements. The resulting chimeric molecules was produced in recombinant form and analysed by its ability to process its substrate Furimazine at different concentrations of Calmodulin Binding Peptide (CaM-BP). As can be seen in Figure 2 the luminescence of the biosensor increased dose-dependently and saturably upon addition of CaM-BP. The overall luminescence change was close to 20 fold that is one of the largest dynamic changes observed in artificial signaling systems (Fig. 2 right panel).
  • CaM-BP Calmodulin Binding Peptide
  • the availability of the CaM-operated switch module encouraged us to apply the earlier developed concept of two component biosensors to construct sensors of rapamycin and tacrolimus.
  • the CaM-NanoLuc module was fused to FKBP and a mutant version of CaM-BP was fused to either FRB (rapamycin biosensor) or to Calcineurin A and Calcineurin B complex fusion by a linker (tacrolimus biosensor) (Fig 3; SEQ ID NO:4).
  • FRB rapamycin biosensor
  • Calcineurin A and Calcineurin B complex fusion by a linker tacrolimus biosensor
  • VHH binders were extracted from PDB structure lbvn.pdb and lkxv.pdb and were fused to the N and C terminus of the cp-NanoLuc to construct and develop an alpha amylase biosensor (PIG. 6).
  • tacrolimus biosensor The main impediment to that is the size of tacrolimus binding ternary complex that is more than two times larger than the PRB:PKBP complex that is used to bind rapamycin (Ligure 7).
  • FIG. 9C shows the result of drying and rehydration that shows that the biosensor can be dried and rehydrated without any optimization of the conditions.
  • FIG. 9D We observed that 50% hypotonically lysed blood significantly diminished the biosensor’s signal. This is not surprising as the emission wavelength of the NanoLuc overlaps with the absorption wavelength of haemoglobin.
  • One way of executing option b) is to co-integrate the biosensor with a luminescent converter that absorbs light in the blue part of the spectrum and emits at a higher wavelength. This phenomenon is termed Bioluminescence Resonance Energy Transfer or BRET.
  • BRET Bioluminescence Resonance Energy Transfer
  • FIG 10B shows the emission scan of such rapamycin biosensor in the absence and presence of rapamycin. It can be seen that the emission of the biosensor, as expected is shifted to the maximum of 5l0nm that corresponds to the emission of EGFP.
  • the results of titration of the red shifted biosensor with rapamycin are shown in Figure 10C and demonstrate that despite the integration of the wavelength“extender” domain the biosensor faithfully produced enhanced light emission with the maximum at 510nm.
  • the signal change could be fitted to a K d of 0.5nM that demonstrates that despite introduction of the BRET-forming domain the function of the biosensor remained largely unchanged.
  • tacrolimus biosensors with either EGFP or a red fluorescent protein such as Cherry or mTomato (FIG. 11 A).
  • a small organic red dye may be conjugated to the NanoLuc using site selective chemistry (FIG. 11B). This may be achieved using thiol chemistry in case cysteines are absent from the sequence of the protein biosensor (or present at positions where they can be mutated to serine without compromising the structure or function of the protein).
  • codon reassignment can be used to incorporate a biorthogonal moiety such as 4-Azidophenylalanine that can be used for dye conjugation via click chemistry.
  • a biorthogonal moiety such as 4-Azidophenylalanine that can be used for dye conjugation via click chemistry.
  • An alternative approach would be to use the recently reported furimazine derivatives with red shifted emission maximum 2 .
  • Such substrates can be used for forming BRET pairs resulting in far-red shifted emission (FIG. 12 bottom panel).
  • the exact quantum yields, stability and compatibility with the developed biosensors are not known and need to be investigated.
  • Fluorescent proteins such as GFP may be utilized in light-emitting biosensors.
  • a single component biosensor comprises first and second GFP fragments.
  • An SH3 domain is N-terminal of the first GFP fragment
  • a calmodulin sensor interconnects the first and second GFP fragments
  • an SFI3 ligand peptide is C-terminal of the second GFP fragment.
  • fluorescence emission is minimal.
  • the first and second GFP fragments interact as a result of the calmodulin sensor binding calmodulin-binding peptide that induces a conformational change of the latter leading to reconstitution of the GFP structure and resulting in increased light emission.
  • the SH3 domain and the SH3-binding peptide act as a stabilizer to facilitate the interaction between first and second GFP fragments.
  • the single-component biosensor of FIG. 15 may be incorporated into a“two- component” biosensor as shown in FIG. 16.
  • the GFP peptide sensor (which is essentially the same as that shown in FIG. 15) of the first molecular component is fused to an FKBP binding moiety.
  • the second molecular component comprises a low affinity derivative of M13 calmodulin binding peptide fused to FRB. Binding of rapamycin target molecule results in increase of the local concentration of both of components and association of the calmodulin binding peptide with calmodulin. The latter switches the conformational change restoring the active conformation of GFP thereby significantly increasing fluorescence emission by the GFP sensor.
  • FIG. 14 An example of another two-component biosensor is shown in FIG. 14.
  • the first component comprises an FKBP sensor fused to a GFP fragment with a C-terminal calmodulin amino acid sequence.
  • the second component comprises an FRB sensor fused to a second GFP fragment with a C-terminal calmodulin-binding peptide.
  • GFP Upon binding rapamycin by FRB and FKBP, GFP is activated by conformation/solvation driven conformation change to thereby increase fluorescence emission.
  • the calmodulin-peptide binding interaction acts as a stabilizer rather than as an allosteric switch.
  • a single component GFP-based biosensor may be produced comprising first and second GFP fragments of a circularly-permuted GFP protein.
  • FKBP is located N-terminally of the first GFP fragment and FRB is located C-terminally of the second GFP fragment.
  • fluorescence emission is low.
  • light emission increases substantially in a dose-dependent manner.
  • the functional mechanism of this biosensor is expected to be similar to the one described for single component NanoLuc biosensor in Figure 6.
  • a domain fused to a b-strand at the end of the reporter protein displaces this strand due to Brownian motion and solvation forces rendering the GFP reporter molecule non-functional.
  • Addition of the ligand constrains the movement of the b- strand and increases the proportion of the functional GFP in the sample. Therefore the dynamic range of the biosensors can be further increased by rigidifying the linker between the protein and the last b-strand.
  • This model provides guidance in further improving the dynamic ranges of biosensors based on fragment displacement.
  • this can be achieved by further increasing the rigidity of the linker connecting the fragment to the enzyme, alternatively this can be achieved by extending the linker thereby increasing the hydrodynamic radius of the fragment-binder assembly and decreasing the frequency of its association with the reporter domain (B).
  • the dynamic range is increased by creating a steric barrier by fusing a disordered peptide or polymer sequence (such as PEG) to the binder thereby creating a barrier for ligand-independent reporter reconstitution.
  • the increase in dynamic range can be achieved by introducing the interactions that stabilise the biosensors conformation in the “open” form where binding of the ligand shifts the equilibrium to the“closed” form.
  • the examples provided herein demonstrate the feasibility of a generic approach for construction of protein switches as outlined in FIG. 19.
  • the gene coding for a protein of interest is used to create a library of calmodulin (CaM) insertion mutants.
  • CaM calmodulin
  • Such library can be either computationally designed using available structural information and placing the calmodulin sequences in the loop regions or it can be constructed by transposon mediated random insertion of CaM sequence in to the sequence of Pol.
  • the library is them expressed either in vivo (bacteria, yeast or eukaryotic cells) or in vitro using cell-free expression and appropriate activity assays are used to identify mutants that are activated or inactivated by calmodulin binding peptide. These variants can be then used to construct two component biosensors.
  • the identified mutants are further analysed and are used to construct circular permutated variants.
  • the identified insertion sides can be then used to construct single component biosensors by adding binding domains to the N and C terminus of the sequence.
  • the calmodulin and calmodulin binding peptide amino acid sequences used in this example may be wild-type amino acid sequences or be variant sequences, such as created by mutagenesis.
  • NanoLuc-CalM peptide sensor SEQ ID NO:l
  • NanoLuc-CalM-FKBP Rosham and FK506 sensor component 2; SEQ ID NO:2
  • SUMO-Cal-alpha/beta-CalM-BP NanoLuc-CalM-FK506 sensor component 1; SEQ ID NO:4
  • VHHl-cpNanoLuc-VHH2 Single component of amylase sensor
  • Single component GFP biosensor comprising SH3 stabilizer (SEQ ID NO:9)
  • FKBP-GFP-FRB FKBP-GFP-FRB
  • VQVETISPG DG RTFPKRGQTCVVHYTG M LEDG KKFDSSRDRIMK PFKFM LG KQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATG H PG I I PPHATLVFDVELLKL
  • FKBP-GFP peptide sensor(SH3L-GFP-CalM-SH3) FKBP-GFP peptide sensor(SH3L-GFP-CalM-SH3) :

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Abstract

L'invention concerne un biocapteur moléculaire comprenant un ou plusieurs capteurs et une ou plusieurs protéines ou fragments de protéines, tels qu'une enzyme ou une protéine fluorescente, capables d'émettre de la lumière, ou de faciliter l'émission de lumière, en réponse au capteur. L'invention concerne en outre des dispositifs de détection comprenant le biocapteur moléculaire. L'invention concerne également des méthodes de détection d'une molécule cible et un diagnostic de maladie utilisant le biocapteur moléculaire.
PCT/AU2019/050367 2018-04-24 2019-04-24 Biocapteurs électroluminescents Ceased WO2019204873A1 (fr)

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CN113336859A (zh) * 2021-05-25 2021-09-03 大连理工大学 一种识别cd105的生物探针
WO2023131871A1 (fr) * 2022-01-05 2023-07-13 Queensland University Of Technology Commutateurs à base de protéine allostérique de porte et
CN119570760A (zh) * 2025-02-05 2025-03-07 北京理工大学 一种荧光传感器蛋白设计方法及其产品和检测方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113336859A (zh) * 2021-05-25 2021-09-03 大连理工大学 一种识别cd105的生物探针
WO2023131871A1 (fr) * 2022-01-05 2023-07-13 Queensland University Of Technology Commutateurs à base de protéine allostérique de porte et
CN119570760A (zh) * 2025-02-05 2025-03-07 北京理工大学 一种荧光传感器蛋白设计方法及其产品和检测方法

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