WO2020207453A1 - 基于CRISPR/Cas12a系统的生物传感器、试剂盒及其在小分子检测中的用途 - Google Patents

基于CRISPR/Cas12a系统的生物传感器、试剂盒及其在小分子检测中的用途 Download PDF

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WO2020207453A1
WO2020207453A1 PCT/CN2020/084111 CN2020084111W WO2020207453A1 WO 2020207453 A1 WO2020207453 A1 WO 2020207453A1 CN 2020084111 W CN2020084111 W CN 2020084111W WO 2020207453 A1 WO2020207453 A1 WO 2020207453A1
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dsdna
crispr
transcription factor
cas12a
probe
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English (en)
French (fr)
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张立新
谭高翼
梁敏东
王为善
刘家坤
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华东理工大学
中国科学院微生物研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of biological detection, and relates to the use of allosteric transcription factor (aTF) and the CRISPR/Cas12a system to convert small molecule signals into light signals through the cis-cutting and trans-cutting activities of CRISPR/Cas12a Biosensors, compositions and kits, as well as methods for in vitro detection of small molecules using them.
  • aTF allosteric transcription factor
  • CRISPR/Cas12a system to convert small molecule signals into light signals through the cis-cutting and trans-cutting activities of CRISPR/Cas12a Biosensors, compositions and kits, as well as methods for in vitro detection of small molecules using them.
  • uric acid is generally produced and discharged in a dynamic balance in the body, and excessive accumulation affects the normal function of human cells.
  • hyperuricemia and gout nephropathy all use uric acid as an indicator, which is a biomarker for detection and plays an important role in the field of disease monitoring.
  • P-Hydroxybenzoic acid (p-HBA) is widely used in food preservatives, drug synthesis and cosmetics because of its inhibitory effect on fungi and bacteria. Once it exceeds the standard, the harm is also huge. Therefore, the development of new small molecule detection methods is essential.
  • the small molecule detection methods in the prior art are mainly divided into two categories: physical and chemical analysis methods and biological analysis methods.
  • the physical and chemical analysis methods are mainly through spectroscopy, chromatography and their combined technology. These technologies have high separation efficiency, good selectivity, and strong qualitative and quantitative capabilities.
  • the sample preparation process is complicated, the instrument is expensive and time-consuming. Even skilled operators need a long time to get results, and it is not suitable for on-site analysis.
  • Bioanalysis methods usually rely on specific interactions between small molecules and proteins/nucleic acids inherent in the body to realize signal generation, amplification, signal conversion and readout on a smaller system or chip. Such a method can realize on-site, real-time detection.
  • the receptor or antibody interacting with the small molecule is usually used to capture small molecules by simulating agonist/antagonist-receptor or antigen-antibody interaction pairs.
  • nucleic acids such as aptamers
  • the signals of such interactions are converted and amplified into light/chromogenic/electrochemical signals to read out qualitative or quantitative information of small molecules.
  • the CRISPR/Cas system is an adaptive immune defense mechanism that bacteria and archaea use Cas protein to resist foreign nucleic acid invasion under the guidance of RNA.
  • a clustered repeating DNA sequence with unknown function was first reported in 1978, and it was officially named Clustered Regularly Interspersed Palindromic Repeats (CRISPR) in 2001.
  • CRISPR Clustered Regularly Interspersed Palindromic Repeats
  • CRISPR/Cas systems are divided into 2 types and 5 types; Type 1 CRISPR/Cas systems use multiple effector protein complexes to interfere with target genes; The Type 2 CRISPR/Cas system uses a single effector protein to resist the invasion of foreign nucleic acids, and has attracted wide attention because of its simplicity and efficiency.
  • Cas9 and Cas12a also known as Cpf1
  • Cpf1 Cas9 and Cas12a
  • the “DETECTR” DNA endonuclease targeted CRISPR transreporter
  • the “HOLMES” an one-HOur Low-cost Multipurpose Highly Efficient System
  • the present invention provides a biosensor for detecting small molecules, the biosensor comprising an identification element and a transducing element, wherein,
  • the recognition element includes an allosteric transcription factor (aTF) and activated double-stranded DNA (activated dsDNA).
  • the activated dsDNA includes the binding site of the aTF and the recognition site of the CRISPR/Cas12a system (ie, the protospace Region proximity motif (PAM) and a sequence that is at least partially complementary to the guide RNA, and
  • the transducing element includes the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the guide RNA (gRNA), and the ssDNA probe is two The ends are respectively conjugated with a luminescent/chromogenic group and its quenching group;
  • the recognition element includes an allosteric transcription factor (aTF) and a double-stranded DNA probe (dsDNA probe), the dsDNA probe includes the binding site of the allosteric transcription factor and the recognition site of the CRISPR/Cas12a system Point PAM and a sequence that is at least partially complementary to the guide RNA (gRNA), and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and its quenching group, and
  • aTF allosteric transcription factor
  • dsDNA probe includes the binding site of the allosteric transcription factor and the recognition site of the CRISPR/Cas12a system Point PAM and a sequence that is at least partially complementary to the guide RNA (gRNA), and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and its quenching group, and
  • the transduction element includes the CRISPR/Cas12a system, and the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the gRNA.
  • the binding of the aTF and the target small molecule makes the binding affinity of the allosteric transcription factor and the activated dsDNA and/or dsDNA probe Increase or decrease, thereby reducing or increasing the free form of activated dsDNA and/or dsDNA probe; the free form of activated dsDNA acts as an activator to form a ternary complex with the CRISPR/Cas12a protein and the gRNA, thereby activating CRISPR
  • the trans-ssDNA cleavage activity of the /Cas12a protein enables the ssDNA probe to be cleaved and generates a detectable light signal, or the free form of the dsDNA probe is recognized by the gRNA and is cis-cut by the CRISPR/Cas12a protein. Cutting is performed to generate a detectable light signal; through the generated light signal, the presence and/or content of the target small analysis can be
  • the present invention provides a kit for detecting small molecules, the kit comprising the biosensor according to the first aspect.
  • the kit further includes a detection element for detecting light signals.
  • the present invention provides a method for detecting small molecules in a sample, the method comprising: contacting the sample with the biosensor of the first aspect to generate a light signal, and detecting The generated light signal is detected, thereby detecting small molecules in the sample.
  • the present invention provides a composition for detecting small molecules, the composition comprising a recognition reagent and a transducing reagent, wherein,
  • the recognition reagent contains allosteric transcription factor (aTF) and activated double-stranded DNA (activated dsDNA).
  • the activated dsDNA contains the binding site of the aTF, the recognition site PAM of the CRISPR/Cas12a system, and the guide RNA At least partially complementary sequences, and
  • the transduction reagent includes the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the guide RNA (gRNA), and the ssDNA probe is two The ends are respectively conjugated with a luminescent/chromogenic group and its quenching group;
  • the recognition reagent includes aTF and a double-stranded DNA probe (dsDNA probe), and the dsDNA probe includes the binding site of the aTF, the recognition site PAM of the CRISPR/Cas12a system, and the guide RNA (gRNA) At least a partially complementary sequence, and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and its quenching group, and
  • the transduction reagent includes the CRISPR/Cas12a system, and the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the gRNA.
  • the present invention provides a kit for detecting small molecules, the kit comprising the composition of the fifth aspect.
  • the present invention provides a method for detecting small molecules, the method comprising contacting the test sample with the composition of the fifth aspect or the kit of the sixth aspect to generate light Signal, and detecting the generated light signal to detect the small molecule.
  • the present invention provides the use of the biosensor of the first aspect, the composition of the fourth aspect, and the kit of the second or fifth aspect in the detection of small molecules.
  • the present invention uses aTF and CRISPR/Cas12a-mediated small molecule detection tools
  • C RISPR/Cas12a-and aT F-mediated s mall m ol e cu l e detect or , referred to as "CaT-SMelor” C RISPR/Cas12a-and aT F-mediated s mall m ol e cu l e detect or , referred to as "CaT-SMelor”
  • the detection method of the present invention is not only easy to operate, time-saving (up to 25 minutes, or even 5-10 minutes) and cost-saving (less than 2 yuan per reaction), and high sensitivity (the detection limit is reduced to nM Grade), high accuracy, and small reaction system (for example, blood samples only need 1 ⁇ L), can be operated on 96-well plates or 384-well plates, so it is suitable for high-throughput and accurate detection for diagnostic and non-diagnostic purposes.
  • Fig. 1 is a schematic diagram of the principle of small molecule detection using the biosensor of the present invention according to an embodiment of the present invention.
  • aTF is immobilized. Specifically, aTF and cellulose domain (CBD) are fused and expressed, and then the fusion protein aTF-CBD is mixed with microcrystalline cellulose (MC) to fix it on MC; the fusion protein aTF-CBD is combined with activated dsDNA to form aTF-CBD-DNA complex; the binding of small molecules to aTF leads to aTF allosteric effect, so that activated dsDNA is separated from aTF, and free form of activated dsDNA (also called "free dsDNA”) is obtained; the free form of activated dsDNA Add the CRISPR/Cas12a system and the ssDNA probe (reporter: FQ-labeled ssDNA), the free activated dsDNA activates the CRISPR
  • the required amount of blood sample used is small ( ⁇ 1 ⁇ L), and the volume of the reaction system is small, which can be operated on 96/384-well plates.
  • Use common instruments such as a microplate reader to detect light signals to achieve qualitative/quantitative analysis of small molecules.
  • Figure 2 illustrates the comparison of the fusion protein CBD-aTF (CBD-HucR, CBD-HosA and CBD-TetR) prepared in Example 1 to Example 2 by gel retardation experiment (EMSA) according to an embodiment of the present invention The binding ability of dsDNA and the dissociation diagram of CBD-aTF-dsDNA in the presence of specific small molecules.
  • Figure 2A Schematic diagram of the structure of the fusion proteins CBD-HosA, CBD-HucR and CBD-TetR.
  • FIGS. 2B-2C Diagrams showing the binding of the fusion protein CBD-HucR to activated dsDNA (dsDNA (HucR) ) in the absence or presence of uric acid (measured by EMSA).
  • Figure 2D-2E A graph showing the binding of the fusion protein CBD-TetR to activated dsDNA (dsDNA (TetR) ) in the absence or presence of tetracycline (measured by EMSA).
  • Figure 2F-2G Diagram showing the binding of the fusion protein CBD-HosA to activated dsDNA (dsDNA (HosA) ) in the absence or presence of p-HBA (measured by EMSA) .
  • FIG. 3 is a graph showing the detection of target small molecules using the biosensor of Example 1 to Example 2 according to an embodiment of the present invention.
  • Fig. 3A shows a flow chart of detecting small molecules using the biosensor of embodiment 1 to embodiment 2 of the present invention.
  • Figure 3B A diagram showing the binding of the fusion protein CBD-HucR to activated dsDNA (dsDNA (HucR) ) in the presence of 500 ⁇ M uric acid and its structural analogs (adenine, guanine, hypoxanthine) (using EMSA measurement).
  • Figure 3C A graph showing the fluorescence intensity detected by the uric acid biosensor of Example 1 of the present invention with 500 ⁇ M uric acid (UA) and its structural analogs (adenine, guanine, hypoxanthine) as samples, Among them, 40nM free activated dsDNA (dsDNA (HucR) ) was used as a positive control (PC).
  • Figure 3D shows the presence of 1.8mM p-hydroxybenzoic acid (p-HBA) and its structural analogs (Tyrosol (Tyrosol, also known as “p-hydroxyphenyl ethanol”), p-aminobenzoic acid (p-ABA) ), methyl p-hydroxybenzoate (p-MHB), p-hydroxybenzyl alcohol (p-HBnOH)), the fusion protein CBD-HosA and activated dsDNA (dsDNA (HosA) ) binding diagram (measured by EMSA ).
  • p-HBA p-hydroxybenzoic acid
  • p-ABA p-aminobenzoic acid
  • p-MHB methyl p-hydroxybenzoate
  • p-HBnOH p-hydroxybenzyl alcohol
  • Figure 3E shows the detection of 1.8mM p-HBA and its structural analogues (Tyrosol, p-ABA, p-MHB, p-HBnOH) as samples, using the p-HBA biosensor of Example 2 of the present invention
  • FIG. 4 is a graph showing the detection of target small molecules using the biosensors of Example 1 to Example 2 of the present invention according to an embodiment of the present invention.
  • Figure 4A The detection results of the effect of different concentrations of activated dsDNA on the change of fluorescence intensity, and the slope of the linear increase interval of fluorescence value is taken, and the concentration of activated dsDNA is used as the abscissa, and the corresponding slope is calculated as the ordinate to indicate the linear detection range of activated dsDNA. The regression equation.
  • Figure 4B Detecting results of the effect of different concentrations of uric acid on the change of fluorescence intensity, and taking the slope of the linear increase interval of the fluorescence value, using the concentration of uric acid as the abscissa and the corresponding slope as the ordinate to calculate the linear detection range of uric acid as 25- 500nM.
  • Figure 4C The detection results of the effect of different concentrations of p-HBA on the change of fluorescence intensity, and the slope of the linear increase interval of the fluorescence value is taken, and the p-HBA concentration is used as the abscissa and the corresponding slope is the ordinate to calculate the p-HBA The linear detection range is 9-180nM.
  • FIG. 5 is a diagram showing the measurement of uric acid concentration in a human blood sample using the uric acid biosensor of Example 1 of the present invention in comparison with traditional uric acid concentration measurement methods (HPLC and Backman kit) according to an embodiment of the present invention chart.
  • Fig. 5A A chart showing the detection of uric acid in a human blood sample using the uric acid biosensor of the present invention and the HPLC method.
  • Fig. 5B A chart showing the detection of uric acid in human blood samples using the uric acid biosensor of the present invention and the Backman kit used clinically.
  • Fig. 5C exemplarily illustrates a flow chart of using the biosensor of the present invention in blood sample analysis.
  • the present invention utilizes CaT-SMelor to change the affinity between aTF and double-stranded DNA and the cis-cutting activity and trans-single-stranded DNA cleavage activity of CRISPR/Cas12a through the combination of target small molecules and aTF. /Or the ssDNA probe is cut to convert the signal of the target small molecule into a corresponding light signal to realize the qualitative detection of the small molecule. Furthermore, the inventors found that for target small analysis, within a certain concentration range, the change in the slope of the generated fluorescence curve is proportional to the concentration of the small molecule. Therefore, the slope of the fluorescence curve of the sample containing the target small molecule was measured. The measurement can achieve accurate quantitative detection of target small molecules in the sample.
  • the present invention provides a composition for detecting small molecules, the composition comprising a recognition reagent and a transduction reagent, wherein,
  • the recognition reagent comprises an allosteric transcription factor (aTF) and double-stranded DNA (dsDNA), and the dsDNA comprises a binding site of the aTF, a recognition site PAM of the CRISPR/Cas12a system, and a sequence that is at least partially complementary to the guide RNA ;
  • the transduction reagent includes the CRISPR/Cas12a system,
  • composition also contains a luminescent/chromogenic group.
  • the transduction reagent further comprises single-stranded DNA (ssDNA).
  • ssDNA single-stranded DNA
  • the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the guide RNA (gRNA).
  • gRNA guide RNA
  • the dsDNA can be used as an activating dsDNA, or dsDNA probe.
  • the ssDNA can also be used as a probe.
  • both ends of the dsDNA probe or ssDNA probe may be conjugated with a luminescent/chromogenic group and its quenching group.
  • the composition of the present invention includes a recognition reagent and a transduction reagent, the recognition reagent comprising an allosteric transcription factor (aTF) and activated double-stranded DNA (activating dsDNA), the activated dsDNA comprising the aTF
  • aTF allosteric transcription factor
  • activating dsDNA activated double-stranded DNA
  • the transduction reagent includes the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the guide RNA (gRNA), and the ssDNA probe is two The ends are respectively conjugated with a luminescent/chromogenic group and its quenching group.
  • the composition of the present invention includes a recognition reagent and a transduction reagent
  • the recognition reagent includes aTF and a double-stranded DNA probe (dsDNA probe)
  • the dsDNA probe includes a binding site of the aTF.
  • Point, the recognition site PAM of the CRISPR/Cas12a system, and a sequence that is at least partially complementary to the guide RNA (gRNA), and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and its quenching group, and
  • the transduction reagent includes the CRISPR/Cas12a system, and the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the gRNA.
  • the composition of the invention may be in the form of a biosensor.
  • Biosensers are generally considered to be a type of device that analyzes biological materials (such as tissues, microbial cells, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.) or biologically derived materials or biological simulation materials.
  • Materials, etc. are closely combined or connected with physical and chemical sensors or sensing micro-systems (which can be optical, electrochemical, thermal, piezoelectric or magnetic), which can generate intermittent or continuous signals (optical signals, electrical Signal, etc.), the signal intensity is proportional to the analyte (for example, a linear relationship), thereby performing the analysis function.
  • a biosensor has a general definition, usually composed of two parts: an identification element (also called a molecular recognition element) and a transducing element (also called a transducer).
  • an identification element also called a molecular recognition element
  • a transducing element also called a transducer
  • the term "biosensor” refers to a device or device that includes identification elements and transduction elements, and is also referred to as a "biosening platform”.
  • the biosensor described herein may include an amplifying element that further improves detection sensitivity and/or a detection element that directly performs detection.
  • the biosensor described herein is not limited to a specific form, as long as any form including the identification element and the transducing element described below is included in the scope of the term "biosensor” herein.
  • the identification element is the key element of the biosensor. It identifies the analyte and undergoes a biological reaction, which directly determines the function and quality of the sensor.
  • the recognition element includes aTF and activating dsDNA, or aTF and dsDNA probe.
  • allosteric transcription factor used in the present invention has a well-known meaning in the art.
  • aTF usually contains a DNA binding domain (DBD) and an effector binding domain (EBD).
  • DBD DNA binding domain
  • EBD effector binding domain
  • the binding of substances changes the binding of aTF to the target gene sequence, thereby regulating the expression of related genes and quickly responding to changes in the outside world.
  • aTF can be divided into different families such as AraC, LacI, TetR, etc. These aTFs can often specifically sense various small molecule compounds and play an important role in maintaining the stability of metabolism.
  • the allosteric transcription factor includes a domain (EBD) that binds to an effector (usually a small molecule) and a DNA binding domain (DBD), and its binding to the effector causes a conformational change, making the allosteric transcription factor and its specificity
  • EBD domain
  • DBD DNA binding domain
  • the binding affinity of the interacting DNA fragment (usually the promoter operating sequence in its natural state) changes, thereby enhancing or weakening the transcription of the DNA sequence controlled by the operating sequence (Nat Methods. 2016, 13(2): 177-183) ). In this way, gene transcription depends on the concentration of small molecules.
  • a transcription factor binding site is also referred to as a transcription factor action site, a transcription factor manipulation site or a transcription factor binding motif.
  • the transcription factor action site is usually complementary double-stranded DNA.
  • a single 3',5'-phosphodiester bond break (gap) near the transcription factor action site does not affect the binding of the allosteric transcription factor to its action site.
  • the sequences of transcription factor sites in the natural transcription system are known. Different species have slightly different lengths of transcription factor binding sites. The average length of transcription factor binding sites in Escherichia coli is 24.5bp, and in Drosophila it is 12.5bp (J Mol Biol. 1998, 284(2): 241-54; Nucleic Acids Res. 2003, 31(1): 374-8). In the present invention, the length of the transcription factor action site is preferably 10-40 bp, more preferably 15-25 bp, and most preferably 17-19 bp.
  • the DNA fragments that interact with allosteric transcription factors are not limited to the sites of action of allosteric transcription factors that exist in natural systems.
  • the equilibrium dissociation constant between the allosteric transcription factor and the DNA fragment that interacts with the allosteric transcription factor can be increased and/or the equilibrium dissociation constant between the allosteric transcription factor and small molecules can be reduced, and further Improve the sensitivity of the sensor of the present invention.
  • the combination of allosteric transcription factors and small molecules can make the allosteric transcription factors and target dsDNA fragments (ie, DNA fragments containing the sites of action of allosteric transcription factors), also referred to herein as "activating dsDNA” or The "dsDNA probe") has an increased binding affinity (activation system) or decreased (repression system).
  • the allosteric transcription factor of the present invention is an allosteric transcription factor that activates the system.
  • the binding of the allosteric transcription factor to the small effector molecule increases its binding affinity to the target dsDNA (that is, the binding of the small molecule enables the allosteric transcription factor to bind to the target dsDNA fragment), thereby making the free target
  • the amount of dsDNA ie, target dsDNA that is not bound to allosteric transcription factors is reduced.
  • the equilibrium dissociation constant of the transcription factor-small molecule complex formed by the binding of the allosteric transcription factor and the small molecule to the target dsDNA fragment is greater than that of the allosteric transcription factor and the target dsDNA fragment
  • the equilibrium dissociation constant of binding; preferably, the equilibrium dissociation constant of the binding of the allosteric transcription factor-small molecule complex to the target dsDNA fragment is the equilibrium dissociation constant of the binding of the allosteric transcription factor to the target dsDNA fragment 10-10000 times, preferably 20-5000 times, more preferably 50-1000 times.
  • the allosteric transcription factor of the present invention is an allosteric transcription factor of the repressor system.
  • the binding of the allosteric transcription factor to the small effector molecule weakens its binding affinity to the target dsDNA (that is, the binding of the small molecule makes the allosteric transcription factor detach from its site of action, and the free target dsDNA Volume increase).
  • the equilibrium dissociation constant of the binding of the allosteric transcription factor to the small molecule is greater than the equilibrium dissociation constant of the binding of the allosteric transcription factor to the target dsDNA fragment; preferably, the allosteric transcription factor and the The equilibrium dissociation constant of the binding of the small molecule is 10-10000 times, preferably 20-5000 times, more preferably the equilibrium dissociation constant of the binding of the allosteric transcription factor and the allosteric transcription factor of the present invention to the target dsDNA fragment. Preferably 50-1000 times.
  • the allosteric transcription factor and target dsDNA are provided as a composition or combined into a complex.
  • the allosteric transcription factor and target dsDNA are provided as separate compounds. It should be particularly pointed out that although only the allosteric transcription factor of the repressor system is used in the specific embodiment of the present invention, it is only illustrative, and based on the teaching of this application, those skilled in the art can use different Allosteric transcription factors (for example, other allosteric transcription factors of repression system or allosteric transcription factors of activating system) implement the present invention.
  • the biosensor, composition, kit and method of the present invention can be used for diagnostic, non-diagnostic, or non-clinical purposes, such as environmental pollution monitoring, food and cosmetic quality control, and disease diagnosis.
  • the small molecule to be detected is an effector that changes the conformation of the allosteric transcription factor.
  • a small molecule is characterized in that it has a molecular weight greater than about 50 Daltons but less than about 5000 Daltons (5kD).
  • the small molecule has a molecular weight of less than 1 kD.
  • small molecules may be, for example, environmental indicators, disease indicators, or health indicators, including but not limited to heavy metal ions, toxins, drugs, metabolites, pollutants, or decomposition products of the foregoing substances.
  • Small molecules can exist in the environment or be of bacterial, fungal, plant or animal origin, or they can be artificially synthesized.
  • effector molecules that do not have corresponding allosteric transcription factors, those skilled in the art can add effector binding domains to DNA binding domains through computer simulation design, or modify the effector binding domains of natural transcription factors , To construct an artificial allosteric transcription factor (Nat Methods. 2016, 13(2): 177–183).
  • the small molecule to be detected can be present in any liquid sample or a solid sample that can be converted into a liquid sample by appropriate operations.
  • the sample may be an environmental sample, such as a sample of groundwater, reclaimed water, seawater, wastewater, and mining waste.
  • the sample may be a biological sample, particularly a sample from a subject, such as one or more of the following samples: blood, serum, plasma, sputum, cerebrospinal fluid, urine, tears, alveolar isolates, pleural fluid , Sac fluid, tissue, saliva. Samples can also come from food, drinking water, cosmetics or feed.
  • the sample can be pretreated to enrich and extract the small molecules to be detected, or to remove impurities that may interfere with the detection. For example, centrifugation, filtration, ultrasound, homogenization, heating, freezing, thawing, mechanical treatment or a combination of multiple operation methods, and/or adding pretreatment reagents.
  • pretreatment reagents include surfactants and detergents, salts, cell lysis agents, anticoagulants, degrading enzymes (such as proteases, lipases, nucleases, lipases, collagenases, cellulases, amylases). Etc.) and solutions (e.g. buffer).
  • Table 1 lists a variety of allosteric transcription factors, the DNA sequences that interact with them, and the corresponding small effector molecules. Those skilled in the art can understand that the allosteric transcription factors used in the present invention are not limited to those listed. In addition, one or more bases of the DNA sequence that interacts with the listed allosteric transcription factors can be replaced, deleted, or added to change the allosteric transcription factor and the DNA that interacts with it. The binding strength of the sequence.
  • Table 1 Exemplary allosteric transcription factors, the DNA sequences that interact with them, and the corresponding effectors
  • the recognition element includes an allosteric transcription factor that can be immobilized on a medium and/or an activating dsDNA or dsDNA probe that can be immobilized on a medium. In some embodiments, the recognition element comprises an allosteric transcription factor that can be immobilized on a medium. In some embodiments, the recognition element comprises an activated dsDNA or dsDNA probe that can be immobilized on a medium. In some embodiments, the recognition element includes an allosteric transcription factor that can be immobilized on a medium and an activating dsDNA or dsDNA probe that can be immobilized on the medium.
  • the term “medium” refers to a substance that supports each element and provides a reaction space for each element to facilitate carrying, transportation, packaging, or handling. Those skilled in the art usually fix the element to be fixed on the medium through immobilization technology, such as adsorption method, covalent bonding method, physical embedding method, cross-linking method and other methods commonly used in this field.
  • the medium include, but are not limited to, nitrocellulose membrane or nylon membrane, affinity column chromatography matrix, magnetic beads, solid filler, microcrystalline cellulose, or commercially available protein immobilization media, and the like.
  • the medium is microcrystalline cellulose.
  • the recognition element of the present invention comprises an allosteric transcription factor that can be immobilized on microcrystalline cellulose.
  • the allosteric transcription factor that can be fixed on microcrystalline cellulose is an allosteric transcription factor expressed in fusion with a cellulose domain (hereinafter referred to as "CBD"), and the fusion protein is combined with The adsorption between microcrystalline cellulose can be fixed on microcrystalline cellulose.
  • CBD cellulose domain
  • the recognition element comprises an allosteric transcription factor expressed with a CBD fusion protein.
  • the present inventors fused and expressed the cellulose domain and allosteric transcription factors to obtain a fusion protein that can be immobilized on microcrystalline cellulose.
  • the structural diagram of such a fusion protein is shown in Figure 2A, combining allosteric transcription factors (such as HucR, HosA, TetR, whose amino acid sequences can be shown in SEQ ID NOs: 1-3, respectively) with CBD (its amino acid sequence) It can be connected with a connector as shown in SEQ ID NO: 4).
  • linker is only to connect aTF and CBD, and its amino acid sequence can be shown in Figure 2A (SEQ ID NO: 12), but it is not limited to this. Those skilled in the art can select other linkers according to actual needs.
  • the inventors have confirmed through experiments that the expression of structural transcription factors in the form of fusion proteins (CBD-HucR, CBD-HosA and CBD-TetR) does not adversely affect their binding ability to activated dsDNA and allosteric activity (as shown in Figure 2B- Shown in Figure 2G). Furthermore, comparing the DNA binding capacity and allosteric activity between the fusion protein and the allosteric transcription factor expressed separately, it was found that there was no significant difference between the two.
  • CBD only serves the purpose of fixing allosteric transcription factors to microcrystalline cellulose, and does not affect the function of allosteric transcription factors.
  • the method of fusion expression and purification of allosteric transcription factor and CBD is well known in the art.
  • the gene encoding the allosteric transcription factor with the amino acid sequence shown in SEQ ID NOs: 1-3 and the gene encoding the cellulose domain with the amino acid sequence shown in SEQ ID NO: 4 are cloned into an expression vector (for example, containing His -Tagged pET23b vector), construct the pET23b-aTF-CBD plasmid; transfer the plasmid into the expression bacteria (E. coli BL21), induce expression to obtain the fusion protein.
  • an expression vector for example, containing His -Tagged pET23b vector
  • the fusion protein includes a purified fusion protein.
  • other elements of the present invention for example, the transducer element or detection element described below
  • modifications or improvements can also be fixed on the medium, and those skilled in the art can make modifications or improvements according to actual needs. These improvements or modifications It is also included in the scope of the present invention.
  • activating dsDNA refers to a DNA fragment containing allosteric transcription factor action site, CRISPR/Cas12a protein recognition site PAM, and nucleotides that are at least partially complementary to gRNA, It is double-stranded at least at the allosteric transcription factor action site, the CRISPR/Cas12a protein recognition site, and the sequence at least partially complementary to the gRNA.
  • the activated dsDNA can be used as an activator to activate the trans-cleavage activity of the CRISPR/Cas12a system. Therefore, it is referred to herein as "activated dsDNA”.
  • the length of the activated dsDNA is preferably 20-80 bp, more preferably 55-65 bp, most preferably 58-60 bp.
  • the definition of the binding site of an allosteric transcription factor is as described above.
  • the CRISPR/Cas12a protein recognition site PAM described herein refers to a sequence that can be recognized by the CRISPR/Cas12a system and thus activates the cleavage activity of the CRISPR/Cas12a system. This sequence is called the protospacer adjacent motif (PAM), which is generally rich Contains T (for CRISPR/Cas12a).
  • PAM protospacer adjacent motif
  • the length of PAM is preferably 3-8 bp, more preferably 3-6 bp, most preferably 4 bp.
  • the PAM sequence is not limited to PAM found in natural systems. It is a technique known in the art to select several candidate sequences and verify the binding ability of PAM and CRISPR/Cas12a through random or targeted mutation of the sequence of the action site, or in combination with computer simulation.
  • the nucleotide sequence and PAM sequence of the allosteric transcription factor action site in the activation dsDNA are as follows (SEQ ID NO: 5-7):
  • the "at least partially complementary" to the gRNA refers to at least 50% or more complementary to the gRNA, such as at least 70% or more complementary, such as 80%, 85%, 90%, 95%, 99% or more. 100% complementary sequence.
  • the sequence at least partially complementary to the gRNA may be before, after, or between the transcription factor binding site and PAM, or at least with the transcription factor binding site and/or PAM. Partially overlap, or located within the binding site of allosteric transcription factors.
  • the allosteric transcription factor binding site is the sequence that is at least partially complementary to the gRNA. Therefore, in a preferred embodiment, the activated dsDNA may only include PAM and allosteric transcription factor binding sites (which contain sequences complementary to gRNA). Therefore, the length of activated dsDNA can be as low as 13 or 14 bp.
  • the allosteric transcription factor action site and the PAM sequence can be separated by several nucleotides, for example, 0-20bp, more preferably 5-10bp, most preferably 6 -9bp.
  • the allosteric transcription factor action site and the PAM sequence may be directly connected, or may overlap partially or completely.
  • the PAM sequence may be located in the site of action of allosteric transcription factors.
  • the "activated dsDNA” has both the allosteric transcription factor action site and the CRISPR/Cas12a protein recognition site PAM, so it can bind to the allosteric transcription factor and the Cas12a protein at the same time.
  • the equilibrium dissociation constant of the allosteric transcription factor binding to the activated dsDNA fragment may be higher, lower or similar to the equilibrium dissociation constant of binding the CRISPR/Cas12a protein to the activated dsDNA fragment.
  • the equilibrium dissociation constant of the binding of the allosteric transcription factor and the activated dsDNA fragment is lower than the equilibrium dissociation constant of the binding of the CRISPR/Cas12a protein to the activated dsDNA fragment, that is, the phase Compared with allosteric transcription factors, the activating DNA fragments bind to the CRISPR/Cas12a protein more easily. Therefore, in order to reduce the interference of the allosteric transcription factor and the CRISPR/Cas12a system on each other's functions, reduce noise and improve the sensitivity, the allosteric transcription factor and the CRISPR/Cas system of the present invention can be provided in separate forms and react in different spaces.
  • the activated dsDNA can be prepared by the following method: for example, using a primer pair (for example, a nucleotide sequence such as SEQ ID NOs) that targets specific allosteric transcription factor action sites and CRISPR/Cas protein recognition sites. : 5-18, 6-19 or 7-20) by annealing (for example, 95°C pre-denaturation for 5 min; 95°C denaturation for 30 seconds, 1°C reduction per cycle, 70 cycles; 25°C storage) to form double-stranded DNA; Or it can also be artificially synthesized, for example, artificially synthesized double-stranded DNA with a nucleotide sequence as shown in SEQ ID NOs: 5-7.
  • the activated dsDNA can also be prepared by other methods known in the art.
  • aTF in order to avoid the competitive binding of aTF and CRISPR/Cas12a protein to activate dsDNA in the same reaction system, aTF can be expressed by fusion with other proteins that do not affect its activity (for example, CBD), or activated in dsDNA.
  • CBD do not affect its activity
  • the action site of allosteric transcription factor overlaps with the PAM sequence.
  • probe or “DNA probe” used interchangeably herein refers to a group that is complementary to gRNA and is cleaved by the CRISPR/Cas12a system and has a group that can generate a detectable light signal after being cleaved (in Also referred to herein as a "marker”) nucleotide sequence.
  • the probe has a double-stranded region, or is a double-stranded DNA probe (both referred to as dsDNA probes), and the double-stranded DNA region contains allosteric transcription factor action sites, CRISPR/Cas12a system
  • the length of the dsDNA probe is, for example, 13-100 bp, more preferably 20-50 bp, most preferably 20-30 bp.
  • the definition of the binding site of an allosteric transcription factor is as described above.
  • the nucleotide sequence part of the dsDNA probe may be the same as the activated dsDNA.
  • the length of the sequence recognizable by gRNA and/or crRNA is preferably 15-70 bp, more preferably 15-30 bp, and most preferably 17-24 bp.
  • the dsDNA probe contains a sequence that is at least 70% complementary to gRNA and/or crRNA, such as 80%, 85%, 90%, 95%, 99% or 100% complementary.
  • the allosteric transcription factor action site and the sequence recognizable by gRNA and/or crRNA can be separated by several nucleotides, for example, 0-20bp, More preferably 5-10 bp, most preferably 6-9 bp.
  • the allosteric transcription factor action site and the sequence recognizable by gRNA and/or crRNA may be directly connected, or may overlap partially or completely.
  • the sequence recognizable by gRNA and/or crRNA may be located in the site of action of allosteric transcription factors.
  • the probe is single-stranded DNA
  • the single-stranded DNA can be any nucleotide sequence, such as a nucleotide sequence of about 10-30 bp, preferably about 20 bp in length, such as SEQ ID The nucleotide sequence shown in NO:8.
  • the activating DNA of the present invention is double-stranded DNA and can be designed to be recognized by gRNA and/or crRNA, according to the intention and principle of the present invention, those skilled in the art can use
  • the activated dsDNA is designed as a double-stranded DNA probe (for example, fluorescently labeled at both ends of the activated DNA) to implement the present invention, and this modification should also be included in the scope of the present invention.
  • CRISPR/Cas system refers to two types of CRISPR/Cas systems, which use a single effector protein to resist the invasion of foreign nucleic acids and can perform gene editing and nucleic acid detection simply and efficiently.
  • CRISPR/Cas12a Cpf1
  • the CRISPR/Cas system usually includes at least CRISPR/Cas protein and guide RNA.
  • CRISPR clustered regularly spaced short palindrome repeats
  • CRISPR is a prokaryotic DNA fragment containing short base sequence repeats. After each repetition is a short piece of "spacer DNA" from the previously exposed bacterial virus or plasmid.
  • Cas is a DNA endonuclease guided by RNA (guide RNA).
  • Guide RNA gRNA or sgRNA
  • gRNA or sgRNA is a specific RNA sequence that guides the CRISPR/Cas protein to recognize and cleave the target nucleic acid molecule, which can be transcribed in vitro or artificially chemically synthesized.
  • the guide RNA can be formed by the hybridization of CRISPR RNA (crRNA) and transactivated crRNA (tracrRNA), or can be provided as a separate continuous RNA.
  • the gRNA specifically binds to the complementary target sequence via the target-specific sequence (e.g., spacer sequence "spacer") in the crRNA portion, while the CRISPR/Cas protein binds itself to PAM, and then Cas nuclease mediates the target nucleic acid (e.g., DNA probe). Needle) cutting.
  • target-specific sequence e.g., spacer sequence "spacer”
  • Cas nuclease mediates the target nucleic acid (e.g., DNA probe). Needle) cutting.
  • CRISPR/Cas12a only crRNA guidance is required, not tracrRNA; and CRISPR/Cas12a not only cuts the DNA double-strand of a specific sequence (dsDNA, which contains a sequence complementary to crRNA is recognized by crRNA) but also Any single-stranded DNA (ssDNA) can be cleaved when activated by a DNA double strand of a specific sequence (such as the activating DNA herein) to form a CRISPR/Cas/dsDNA ternary complex.
  • dsDNA which contains a sequence complementary to crRNA is recognized by crRNA
  • ssDNA single-stranded DNA
  • Cas12a itself cannot be used to detect small molecule compounds in various fields such as disease diagnosis, drug development, food safety, and environmental monitoring.
  • the inventors use the recognition element (allosteric transcription factor-activating dsDNA complex), when the PAM on the dsDNA is activated and the CRISPR/Cas protein is activated to activate CRISPR/Cas to cut the probe (target nucleic acid), thereby A detectable light signal is generated to realize the detection of small molecule compounds.
  • the length of the gRNA is preferably 20-70 bp, more preferably 30-50 bp, most preferably 38-45 bp.
  • gRNA can be designed based on the needs of users. It is preferably designed in combination with bioinformatics software.
  • the light signal includes a fluorescent signal or an absorption light signal. Those skilled in the art should understand that in order to quantify the color change that occurs in the analysis, light of a specific wavelength is usually used for excitation and then the absorbed light intensity is detected. Therefore, in the present invention, the term "absorbed light signal" can also refer to the resulting color change (colorimetric analysis).
  • the probe may carry a label to generate a detectable light signal after being cleaved by CRISPR/Cas.
  • labels include but are not limited to: light-emitting organic compounds (such as fluorescein, carotenoids), light-emitting inorganic compounds (such as chemical dyes), fluorophores (such as FAM fluorophores), etc.; nanoparticles and quantum dots, etc.; or chromogenic Mission etc.
  • the technique of labeling nucleic acid sequences with the above-mentioned substances to generate detectable optical signals is well known to those skilled in the art, and can be selected and improved according to actual needs, which does not limit the present invention.
  • the optical signal is a fluorescent signal.
  • the labels are a luminophore and a quencher, which are respectively labeled on the two ends (3' end or 5'end) of the probe.
  • the luminophore is a FAM fluorophore
  • the quencher is a BHQ fluorescence quencher.
  • the optical signal is an absorption optical signal.
  • the label is a chromophore and a quencher group, which are respectively labeled on the two ends (3' end or 5'end) of the probe.
  • the biosensor of the present invention or the composition of the present invention is used to detect small molecules in a sample.
  • the small molecules in the sample can be qualitatively or quantitatively detected.
  • the present invention provides a method for detecting small molecules in a sample to be tested, the method comprising:
  • the present invention provides a method for detecting small molecules in a sample to be tested, the method comprising:
  • the present invention provides a method for detecting small molecules in a sample to be tested, the method comprising:
  • the present invention provides a method for detecting small molecules in a sample to be tested, the method comprising:
  • step (3) Add CRISPR/Cas12a protein and guide RNA to the free dsDNA probe separated in step (2), and detect the generated light signal;
  • the specific conditions for mixing and incubation in step (1) can be selected by those skilled in the art according to actual needs, as long as it is ensured that the allosteric transcription factor and the activating dsDNA or dsDNA probe can fully combine into a complex and the specific conditions in the sample to be tested
  • the small molecule compound can fully bind to the allosteric transcription factor-activating dsDNA or dsDNA probe complex, for example, react at room temperature for more than 1 minute, such as 15-20 minutes (but not limited to this).
  • the ratio of allosteric transcription factor to activated dsDNA or dsDNA probe can be adjusted according to actual needs.
  • the molar ratio of the allosteric transcription factor to the activated dsDNA or dsDNA probe is ⁇ 5:1.
  • the allosteric transcription factor can be completely combined with the activated dsDNA or dsDNA probe to form a protein. -DNA complex.
  • the allosteric transcription factor is an allosteric transcription factor of the repressor system (ie, when a small molecule binds to the allosteric transcription factor, the binding affinity of the allosteric transcription factor to the target dsDNA is weakened, so that the target dsDNA is removed from The aTF-DNA complex breaks away), when the sample to be tested is added, the free dsDNA content increases. Therefore, in a preferred embodiment, in order to reduce background noise and improve the sensitivity of the present invention, the allosteric transcription factor is mixed with dsDNA to form aTF-dsDNA complex, then unbound dsDNA is removed, and then the sample to be tested is added.
  • the allosteric transcription factor is mixed with dsDNA to form aTF-dsDNA complex, then unbound dsDNA is removed, and then the sample to be tested is added.
  • the free dsDNA after adding the test sample directly reflects The presence or content of small molecules in the sample to be tested.
  • the allosteric transcription factor is an allosteric transcription factor that activates the system (that is, when a small molecule binds to the allosteric transcription factor, the binding affinity of the allosteric transcription factor and dsDNA is enhanced, so that dsDNA and other The constitutive transcription factor binds to more aTF-dsDNA complexes).
  • the free dsDNA content decreases.
  • the amount of dsDNA added should be determined before step (1), and the calculated reduced free dsDNA indirectly reflects the presence or content of small molecules in the sample to be tested .
  • the test sample can be added before, at the same time, or after mixing the dsDNA with the allosteric transcription factor.
  • step (1) may further include adding a medium.
  • the allosteric transcription factor that can be immobilized on the medium is an allosteric transcription factor expressed in fusion with the cellulose domain, the medium is microcrystalline. Cellulose.
  • the allosteric transcription factor is combined with the medium in advance, and the allosteric transcription factor is fixed on the medium to facilitate subsequent operations.
  • a commercially available medium such as microcrystalline cellulose
  • an allosteric transcription factor such as an allosteric transcription factor fused with a cellulose domain
  • the specific conditions can be selected by those skilled in the art according to actual needs, as long as it is ensured that the allosteric transcription factor that can be immobilized on the medium can be sufficiently immobilized on the medium, such as reaction at room temperature for 5-15 minutes (but not limited to this).
  • the unfixed allosteric transcription factor is removed before adding the test sample and dsDNA.
  • the content or concentration of the allosteric transcription factor immobilized on the medium can be determined, for example, using a protein concentration detection method known in the art (such as using a Bradford analysis kit).
  • the method for separating free dsDNA from the protein, small molecule compound, and nucleic acid mixture in step (1) in step (2) is well-known in the art, including but not limited to centrifugation, sedimentation, magnetic bead method, chromatography, affinity And the column method and so on. Those skilled in the art can select specific separation methods and parameters according to actual conditions, which does not limit the present invention.
  • the allosteric transcription factor is an allosteric transcription factor that can be immobilized on a medium
  • free dsDNA fragments can be separated only by filtration or centrifugation (for example, at room temperature, 7000 rpm), which is simple to operate, saves time and Cost is economically feasible. It is also possible to omit the separation step in step (2), and directly add the CRISPR/Cas12a system and optional ssDNA probe to the mixture in step (1).
  • step (3) methods and instruments commonly used in the art for detecting fluorescent signals or absorbing light signals are used to detect the generated light signals.
  • the generated signal is a fluorescent signal
  • a microplate reader is used to measure it.
  • the color change (as mentioned above, included in the range of "absorbed light signal") may not be quantitatively analyzed, but only Colorimetric analysis is also within the scope of the present invention.
  • the generated signal can be analyzed based on the reference level, so as to detect the presence or content of the small molecule.
  • the "reference level” is used interchangeably with “reference sample” and “reference level” in the present invention, and refers to the control of conditions.
  • the reference level may be the level of a sample that does not contain the small molecule.
  • the reference level may be the level of a sample containing a known amount of small molecules.
  • the reference level is a reference value that can normalize the sample to an appropriate standard to infer the presence, absence or content of small molecules in the sample.
  • the reference level may be a previously determined level, for example, a predetermined amount or ratio, and does not need to be determined in the same physical iteration of the detection method described herein.
  • the sample and the reagent may be in the form of a solution, as long as the solution does not affect the binding of aTF to the effector/dsDNA and the cleavage of CRISPR/Cas12a.
  • the solution does not affect the binding of aTF to the effector/dsDNA and the cleavage of CRISPR/Cas12a.
  • the present invention also provides a kit for detecting small molecules, the kit comprising the biosensor and the detection element of the present invention.
  • the detection element refers to an element, device or system that detects the optical signal generated by the biosensor of the present invention.
  • the detection element is used to analyze the light signal generated by the DNA probe by fluorescence analysis or absorption light analysis (including colorimetric analysis), so as to obtain qualitative or quantitative detection results for small molecules.
  • the present invention also provides a kit for detecting small molecules, the kit comprising the reagent of the present invention and a detection reagent.
  • the detection reagent refers to a reagent that detects the light signal generated by using the reagent of the present invention.
  • the detection reagent is used to analyze the light signal generated by the DNA probe by fluorescence analysis or absorption light analysis (including colorimetric analysis), so as to obtain qualitative or quantitative detection results for small molecules.
  • the kit of the present invention further comprises a delivery tool or device (such as a pipette) that uses the biosensor and/or reagent of the present invention to detect small molecules, washing buffer, dilution buffer, and termination buffer.
  • a delivery tool or device such as a pipette
  • Liquid for example, to stop the color development
  • microtiter plate for example, 98-well or 384-well, for reaction and detection
  • one or more containers for example, a data carrier with instructions for use (for example, instructions or computer-readable media ), standards (such as a sample containing a known amount of small molecules), and their combination, etc.
  • composition for detecting small molecules comprising a recognition reagent and a transduction reagent, wherein,
  • the recognition reagent includes allosteric transcription factor (aTF) and double-stranded DNA (dsDNA); and
  • the transduction reagent comprises a CRISPR/Cas12a system, wherein the CRISPR/Cas12a system has a guide RNA,
  • the dsDNA includes a binding site of the aTF, a recognition site PAM of the CRISPR/Cas12a system, and a sequence that is at least partially complementary to the guide RNA,
  • the composition further contains a luminescent/chromogenic group, and the luminescent/chromogenic group is connected to a nucleic acid that can be cleaved by the CRISPR/Cas12a system.
  • composition according to paragraph 1 wherein the transduction reagent further comprises single-stranded DNA (ssDNA).
  • ssDNA single-stranded DNA
  • composition of paragraph 1 or 2 wherein the CRISPR/Cas12a system comprises the CRISPR/Cas12a protein and the guide RNA (gRNA).
  • gRNA guide RNA
  • composition according to paragraph 2 wherein the ssDNA is a ssDNA probe and has a luminescent/chromogenic group and its quenching group.
  • composition according to paragraph 5 or 6 wherein both ends of the dsDNA probe or the ssDNA probe are conjugated with a luminescent/chromogenic group and its quenching group.
  • composition according to any of paragraphs 1-7, wherein the small molecule has a molecular weight of 50-5000 Daltons, preferably 50-1000 Daltons.
  • composition according to any of paragraphs 1-9 wherein the presence of the small molecule triggers the cleavage activity of CRISPR/Cas12a through the aTF and the dsDNA.
  • composition according to any of paragraphs 1-10, wherein the length of the transcription factor binding site is 10-40 bp, preferably 15-25 bp, more preferably 17-19 bp.
  • composition according to paragraph 13 wherein the recognition reagent comprises an allosteric transcription factor that can be immobilized on microcrystalline cellulose and expressed in fusion with a microcrystalline cellulose domain.
  • composition according to any of paragraphs 6-17, wherein the ssDNA probe is any single-stranded DNA of 10-30 bp in length
  • composition of any of paragraphs 6-17, wherein the ssDNA probe is any single-stranded DNA with a length of 20 bp.
  • composition of paragraph 1 wherein the composition comprises a recognition reagent and a transduction reagent, wherein:
  • the recognition reagent includes an allosteric transcription factor (aTF) and activated double-stranded DNA (activated dsDNA), and the activated dsDNA includes a binding site of the aTF, a recognition site PAM of the CRISPR/Cas12a system, and at least part of a guide RNA Complementary sequences, and
  • the transduction reagent includes the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the guide RNA (gRNA), and the ssDNA probe is two The ends are respectively conjugated with a luminescent/chromogenic group and its quenching group;
  • composition of paragraph 1 wherein the composition comprises a recognition reagent and a transduction reagent, wherein:
  • the recognition reagent includes aTF and a double-stranded DNA probe (dsDNA probe), and the dsDNA probe includes a binding site of the aTF, a recognition site PAM of the CRISPR/Cas12a system, and at least part of a guide RNA (gRNA) Complementary sequences, and the dsDNA probes are respectively conjugated with luminescent/chromogenic groups and their quenching groups at both ends, and
  • gRNA guide RNA
  • the transduction reagent includes the CRISPR/Cas12a system, and the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the gRNA.
  • composition of paragraph 1, wherein the composition is in the form of a biosensor.
  • composition of paragraph 23, wherein the biosensor comprises an identification element and a transducing element, wherein
  • the recognition element includes an allosteric transcription factor (aTF) and double-stranded DNA (dsDNA), and the dsDNA includes a binding site of the aTF, a recognition site PAM of the CRISPR/Cas12a system, and a sequence that is at least partially complementary to the guide RNA ;
  • the transduction element includes the CRISPR/Cas12a system,
  • the biosensor also contains a luminescent/chromogenic group.
  • transducing element further comprises single-stranded DNA (ssDNA).
  • composition of paragraph 24 or 25, wherein the CRISPR/Cas12a system comprises the CRISPR/Cas12a protein and the guide RNA (gRNA).
  • the recognition element comprises an allosteric transcription factor (aTF) and activated double-stranded DNA (activated dsDNA), and the activated dsDNA comprises a binding site of the aTF, CRISPR/ The recognition site of the Cas12a system (ie, the adjacent motif of the prototype spacer, PAM) and a sequence that is at least partially complementary to the guide RNA, and
  • the transducing element includes the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the guide RNA (gRNA), and the ssDNA probe is two The ends are respectively conjugated with a luminescent/chromogenic group and its quenching group;
  • the recognition element comprises an allosteric transcription factor (aTF) and a double-stranded DNA probe (dsDNA probe), and the dsDNA probe comprises an allosteric transcription factor
  • aTF allosteric transcription factor
  • dsDNA probe double-stranded DNA probe
  • the binding site, the recognition site PAM of the CRISPR/Cas12a system, and a sequence complementary to at least part of the guide RNA (gRNA), and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and its quenching group Regiment, and
  • the transduction element includes the CRISPR/Cas12a system, and the CRISPR/Cas12a system includes the CRISPR/Cas12a protein and the gRNA.
  • the allosteric transcription factor and dsDNA activation, the CRISPR/Cas12a protein, guide RNA (gRNA) and ssDNA probe are provided as separate reagents.
  • the allosteric transcription factor and dsDNA probe, the CRISPR/Cas12a protein and the guide RNA (gRNA) are provided as separate reagents.
  • kits for detecting small molecules comprising the composition as described in any of paragraphs 1-35.
  • kit further comprises a detection device for detecting light signals.
  • kit according to paragraph 38 or 39, wherein the kit further comprises a delivery tool or device used when operating the biosensor according to any one of paragraphs 1-35, washing buffer, and dilution buffer , Stop buffer, data carrier, standard or container with instructions for use, and their combination.
  • a method for detecting small molecules in a sample to be tested comprising using the composition described in any one of paragraphs 1-35 or the kit described in any one of paragraphs 36-38 to Small molecules in the sample to be tested are detected.
  • step (3) Add CRISPR/Cas12a protein and guide RNA to the free dsDNA probe separated in step (2), and detect the generated light signal;
  • step (1) the molar ratio of the allosteric transcription factor and the activated dsDNA is ⁇ 5:1; the allosteric transcription factor and the The molar ratio of dsDNA probes is ⁇ 5:1.
  • step (1) the allosteric transcription factor and the activated dsDNA are mixed to form a complex, and then the unbound activated DNA is removed, and then added The sample to be tested; or, first mix the allosteric transcription factor and the dsDNA probe to form a complex, then remove the unbound dsDNA probe, and then add the sample to be tested.
  • step (1) the mixing is at room temperature for more than 1 min.
  • step (1) the allosteric transcription factor is an allosteric transcription factor that can be immobilized on a medium.
  • step (1) a medium is added to fix the allosteric transcription factor that can be immobilized on the medium to the medium.
  • sample to be tested is selected from one or more of the following: groundwater, reclaimed water, seawater, wastewater, mining waste; blood, serum, plasma, sputum, cerebrospinal fluid , Urine, tears, alveolar separation, pleural fluid, cyst fluid, tissue, saliva.
  • step (2) separation is performed by filtration, centrifugation, sedimentation, magnetic bead method, chromatography, and affinity column method.
  • step (3) detection is performed by fluorescence analysis or colorimetric analysis.
  • step (4) the optical signal is analyzed based on a reference level, and the reference level is that without the small molecule The level of the sample or the level of a sample containing a known amount of small molecules.
  • composition described in any of paragraphs 1-35 in preparing a kit for detecting small molecules.
  • kit is used for environmental pollution monitoring, food and cosmetic quality control, and disease diagnosis.
  • the dsDNA fragments (as shown in SEQ ID NOs: 5-7), guide RNA (as shown in SEQ ID NOs: 9-11), primers used in the following examples (Shown) and probes (such as the nucleotide sequence shown in SEQ ID NO: 8, with FAM at the 5'end and BHQ at the 3'end) are artificially synthesized by GenScript.
  • LB medium 1% NaCl, 1% peptone, 0.5% yeast powder
  • LB plate 1% NaCl, 1% peptone, 0.5% yeast powder, 1.5% agar powder
  • Binding buffer 50mM Tris-HCl (7.4), 500mM NaCl, 20mM imidazole, 2mM DTT, 5% glycerol
  • Washing buffer 50mM Tris-HCl(7.4), 500mM NaCl, 40mM imidazole, 2mM DTT, 5% glycerol
  • Elution buffer 50mM Tris-HCl(7.4), 500mM NaCl, 500mM imidazole, 2mM DTT, 5% glycerol
  • Dialysis buffer 50mM Tris-HCl (7.4), 500mM NaCl, 2mM DTT, 5% glycerol
  • Ion exchange balance buffer 50mM Tris-HCl(7.4), 2mM DTT, 5% glycerol
  • Ion exchange elution buffer 50mM Tris-HCl(7.4), 1M NaCl, 2mM DTT, 5% glycerol
  • Tris-HCl buffer 50mM Tris-HCl(7.4), 200mM NaCl
  • the allosteric transcription factor HucR and the activating dsDNA fragment (dsDNA hucR ; among them, the HucR binding site (sequence complementary to the gRNA is included) and the CRISPR/Cas12a recognition site (PAM)) are used as recognition Elements, using CRISPR/Cas12a, gRNA and single-stranded DNA (ssDNA; both ends labeled with FAM fluorophore and BHQ fluorescence quenching group as probes) as transducer elements to construct a biosensor platform capable of sensing uric acid.
  • the HucR protein is a repressor protein derived from Deinococcus radiodurans, and its amino acid sequence can be shown in SEQ ID NO:1.
  • HucR in the absence of uric acid (UA), HucR can bind to the HucR site on dsDNA hucR to form a HucR-dsDNA hucR complex.
  • uric acid specifically binds to HucR, so that dsDNA hucR is released from HucR to obtain free form of dsDNA hucR .
  • the free form of dsDNA hucR activates the CRISPR/Cas12a system, causing DNA probe cleavage and generating a fluorescent signal. Therefore, the concentration of uric acid in the sample can be determined by monitoring the fluorescence signal.
  • the CBD fragment was amplified by primer CBD-F/HucR-CBD-R;
  • Use Xho I single enzyme digestion (reaction system: 10x cutsmart buffer 2 ⁇ L; plasmid 1 ⁇ g; Xho I 1 ⁇ L; H 2 O supplemented to 20 ⁇ L; reaction at 37°C for 1 h) to pET23b-HucR plasmid (pre-construction: use Nde I and xho I pair pET23b plasmid and HucR gene fragments were digested with double enzymes (reaction system: 10x cutsmart buffer 2 ⁇ L; plasmid/HucR gene fragments 1 ⁇ g; Xho I 1 ⁇ L; Nde I 1 ⁇ L; H 2 O supplemented to 20 ⁇ L; 37°C reaction for
  • the lysate stream was passed on a HisTrap FF column (GE Healthcare) and eluted with an elution buffer.
  • the peak fraction was collected and dialysis (dialysis membrane from Shanghai Shenggong; dialysis buffer).
  • the dialyzed solution was then loaded onto a HiTrap QHP column (GE Healthcare), and eluted with an elution buffer.
  • the peak fraction was collected and concentrated using a milipore tube.
  • the concentrate was loaded onto a HiLoad 16/600 Superdex 200pg column for fast protein liquid chromatography (FPLC; AKTA Exporer 100, GE Healthcare).
  • the purified HucR-CBD protein was verified by SDS-PAGE and its concentration was determined to be 3 mg/mL using Bradford method.
  • the gel retardation experiment was used to analyze the binding of HucR-CBD and dsDNA HucR .
  • the primers dsDNA HucR -1 and dsDNA HucR -2 were annealed (pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, 75 cycles, with a decrease of 1°C in each cycle) to obtain 60bp dsDNA (HucR) for EMSA analysis.
  • EMSA The experimental conditions and data collection methods of EMSA are based on Wang et al., Molecular Microbiology, 2011, 82(1): 236-250.
  • Mix 0, 25, 50, 100, 200 nM HucR-CBD with 40 nM dsDNA HucR (in a 20 mL buffer: 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM EDTA, 0.1 mM DTT, 5% v/v glycerol, 0.01mg/ml bovine serum albumin, the combination of dsDNA and aTF, and the effector's interaction with dsDNA and aTF are all carried out in this buffer), react for 15 minutes at room temperature in the dark, 1.5% agarose Gel electrophoresis to detect the binding of the fusion protein HucR-CBD and dsDNA HucR .
  • the HucR-CBD: dsDNA HucR 5:1 incubation system was added with different concentrations of uric acid (0, 0.05, 0.5, 5, 50, 500 ⁇ M), the dissociation of dsDNA HucR was detected by EMSA. The results are shown in Figure 2C. As the concentration of uric acid increases, the free dsDNA HucR continues to increase.
  • microcrystalline cellulose Take 2mg of microcrystalline cellulose (Shanghai Shenggong; Item No. 9004-34-6) and wash it twice with Tris-HCl buffer, and mix it with the fusion protein HucR-CBD (200nM) in NEB Buffer, mix and incubate at room temperature for 10 minutes. Discard the supernatant after centrifugation at room temperature and 7000 rpm and use NEB Wash 3 times with buffer to remove unbound protein, use Bradford analysis kit (Tiangen Biochemical Technology Co., Ltd.) to quantify the immobilized protein; add 100nM dsDNA HucR , react at room temperature for ⁇ 9 minutes, centrifuge at room temperature, 7000rpm and discard the supernatant.
  • the supernatant (which may contain free form of dsDNA HucR , with 40nM free form of dsDNA HucR as a positive control) was added to the mixture of Cas12a, gRNA, and FAM/BHQ modified ssDNA (sequence shown in SEQ ID NO: 9) (50nM Cas12a, 50nM gRNA and 250nM ssDNA, in 20 ⁇ L NEB Buffer solution), immediately place the mixture in a BMG CLARIOstar microplate reader (BMG Labtech, UK) for reaction at 37°C, measure and record the fluorescence intensity of the reaction system under 480nm excitation light and 520nm emission light. The results are shown in Figure 3C.
  • the positive control ie free form of dsDNA HucR
  • the uric acid group obtained the strongest fluorescence intensity
  • the uric acid analogs adenine, guanine, hypoxanthine
  • the result is similar to the result of EMSA ( Figure 3B), indicating that the uric acid biosensor of the present invention can accurately identify and detect small molecules.
  • the fluorescence intensity of the reaction system increases.
  • the slope of the linear increase interval of the fluorescence value is taken, and the dsDNA concentration is taken as the abscissa and the corresponding slope is the ordinate as shown in Figure 4A (right column).
  • the linear detection range is 1-25pM.
  • the detection limit of the sensor of this embodiment for uric acid is nM, while the existing uric acid biosensor is usually ⁇ M (such as the uric acid biosensor described in the patent 201810224843.8, 1.71 ⁇ M), which shows that the biosensor of the present invention has excellent detection Sensitivity (can be as low as 25nM).
  • the uric acid biosensor of this embodiment is used to detect uric acid in the serum sample.
  • the supernatant of the serum sample was diluted 10 times with water, and 1 ⁇ L of the diluent was used for detection. Since HPLC can detect the content of uric acid in the serum very accurately, the sample is subjected to HPLC as a control.
  • the detection method of this embodiment is compared with the Backman uric acid detection kit used clinically, and the uric acid concentration measured by Backman is also taken as the ordinate, and the uric acid concentration obtained by the biosensor detection method of this embodiment is taken as the ordinate.
  • the normal range of uric acid in human serum is 166.4 ⁇ M-546.7 ⁇ M, and because the biosensor of the present invention is very sensitive, only a very small amount of blood sample (about 1 ⁇ L) is needed to obtain accurate results (Figure 5C), making the reaction system volume Smaller, the test can be performed on 96-well plates or even 384-well plates.
  • the method of the present invention only takes 15-25 minutes, and results can be obtained in 25 minutes at most, which not only saves time, but also has low cost and simple operation. It is very suitable for high-throughput detection of uric acid in blood and other samples.
  • RT-qPCR real-time fluorescent quantitative PCR
  • RCA rolling circle amplification
  • RPA recombinase polymerase amplification
  • PGM blood glucose meter
  • ISDA isothermal chain displacement amplification
  • clinical method Backman kit
  • CaT-SMelor this Invented biosensor
  • Y applicable
  • N not applicable
  • NA not obtained.
  • the fluorescence half-life of the biosensor exceeds 200 minutes, which provides researchers or other users with more time to record and analyze experimental results.
  • the uric acid biosensor of the present invention has excellent detection sensitivity, high reliability, time and cost saving, simple operation, and is very suitable for in vitro detection of small molecules that require high throughput, high speed and high sensitivity. It has broad application prospects in laboratories, medical and industrial applications.
  • Example 1 In order to verify the universal applicability of the sensor and method of the present invention, the concept of Example 1 was adopted to design sensors that respond to tetracycline and p-HBA respectively.
  • the biosensor that responds to tetracycline uses allosteric transcription factor TetR and double-stranded activating DNA fragments (dsDNA TetR ; which contains TetR binding sites and CRISPR/Cas12a recognition sites (PAM)) as recognition elements, with CRISPR/Cas12a, gRNA and Single-stranded DNA (ssDNA; both ends are labeled with FAM fluorophore and BHQ fluorescence quenching group, as a probe) as the transducer element.
  • dsDNA TetR which contains TetR binding sites and CRISPR/Cas12a recognition sites (PAM)
  • ssDNA Single-stranded DNA
  • the biosensor responding to p-HBA uses the allosteric transcription factor HosA and double-stranded activating DNA fragments (dsDNA HosA ; which contains the HosA binding site and the CRISPR/Cas12a recognition site (PAM)) as recognition elements, and uses CRISPR/Cas12a, gRNA and single-stranded DNA (ssDNA; both ends are labeled with FAM fluorophore and BHQ fluorescence quenching group, as probes) as transducer elements.
  • dsDNA HosA which contains the HosA binding site and the CRISPR/Cas12a recognition site (PAM)
  • ssDNA single-stranded DNA
  • ssDNA both ends are labeled with FAM fluorophore and BHQ fluorescence quenching group, as probes
  • Example 1 According to the method and steps of Example 1, prepare TetR-CBD fusion protein and HosA-CBD fusion protein, construct tetracycline biosensor and p-HBA sensor, and analyze the performance of p-HBA sensor.
  • microcrystalline cellulose Take 2mg of microcrystalline cellulose (Shanghai Shenggong; Item No. 9004-34-6) and wash it twice with Tris-HCl buffer, and mix it with the fusion protein HosA-CBD (200nM) in NEB Buffer, mix and incubate at room temperature for 10 minutes.
  • the supernatant (which may contain free form of dsDNA HosA , at 1.8 mM free form of dsDNA HosA as a positive control) was added to a mixture (50nM) of Cas12a, gRNA (nucleotide sequence shown in SEQ ID NO: 10) and FAM/BHQ ssDNA (sequence shown in SEQ ID NO: 9) Cas12a, 50nM gRNA and 250nM ssDNA, in 20 ⁇ L NEB Buffer solution), immediately place the mixture in a BMG CLARIOstar microplate reader (BMG Labtech, UK) for reaction at 37°C, measure and record the fluorescence intensity of the reaction system under 480nm excitation light and 520nm emission light.
  • BMG CLARIOstar microplate reader BMG Labtech, UK
  • the senor and method of the present invention have universal applicability, and can use a variety of allosteric transcription factors to detect specific small molecule compounds with high sensitivity, high speed and high throughput. It is not only simple to operate, but also low in cost. It has broad application prospects in industrial applications.

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Abstract

本发明涉及包含别构转录因子和CRISPR/Cas12a系统的生物传感器、组合物、试剂盒及其相关的方法和用途。所述生物传感器的机理在于，采用别构转录因子与CRISPR/Cas12a介导的小分子检测工具，通过目标小分子与别构转录因子的结合使得别构转录因子与双链DNA的亲和力发生变化以及采用CRISPR/Cas12a的顺式切割活性和/或反式单链DNA切割活性，对dsDNA探针和/或ssDNA探针进行切割，将目标小分子信号转变为相应的光信号。本发明的生物传感器、组合物、试剂盒及其相关的方法在体外检测小分子时不仅能节约时间和成本，而且灵敏度高并具有高通量检测的潜力。

Description

基于CRISPR/Cas12a系统的生物传感器、试剂盒及其在小分子检测中的用途
相关申请的交叉引用
本申请要求于2019年04月10日向中国国家知识产权局提交的第201910285607.1号中国专利申请的优先权和权益，所述申请公开的内容通过引用整体并入本文中。
技术领域
本发明属于生物检测领域，涉及借助于别构转录因子(allosteric transcription factor，aTF)与CRISPR/Cas12a系统，通过CRISPR/Cas12a的顺式切割切割和反式切割活性将小分子信号转化为光信号的生物传感器、组合物和试剂盒，以及使用其的体外(in vitro)检测小分子的方法。
背景技术
小分子检测之所以如此重要，主要体现在临床诊断、药物研发、食品安全以及环境监测等各个领域的应用。例如，尿酸作为嘌呤代谢终产物，一般在机体内的生成与排出处于动态平衡，过多积累则影响人体细胞的正常功能。其中，高尿酸血症和痛风肾病等都是以尿酸作为指标，是检测方面的生物标志物，在疾病监测领域发挥重要作用。对羟基苯甲酸(p-HBA)因其在真菌及细菌方面的抑制作用，广泛应用于食品防腐剂、药物合成及化妆品领域的使用，一旦超标危害同样巨大。因此开发新的小分子检测方法至关重要。
对样品中小分子的有效检测，在环境污染监控、食品质量控制和疾病诊断等领域都极为重要。现有技术中的小分子检测方法主要分为两大类：理化分析方法和生物分析方法。理化分析方法主要通过波谱法、色谱法及其联用技术。这些技术分离效能高、选择性好、定性和定量能力强。然而，其样品制备过程复杂，仪器价格昂贵，耗时长。即便熟练的操作人员也需要较长时间才能得到结果，且不适用于现场分析。
生物分析方法通常借助体内固有的小分子与蛋白/核酸的特异性相互作用，在较小的体系或芯片上实现信号的生成、放大、信号转换和读出。这样的方法能够实现现场、实时检测。特别地，通常通过模拟激动剂/拮抗剂-受体、或者抗原-抗体相互作用对，利用与小分子相互作用的受体或抗体对小分子进行抓取。或者，通过筛选或进化获得能够与小分子特异性相互作用的核酸(例如适体)，实现对小分子的特异性识别。随后，通过抗体级联放大、石英晶体微量天平等方式，将此类相互作用的信号转化并放大为光/显色/电化学信号，从而读出小分子的定性或定量的信息。
为了扩大能够分析的小分子的范围，已经提出利用别构转录因子与其靶DNA片段以及效应物小分子的相互作用，将小分子的有无或浓度等信息转换为别构转录因子-靶DNA-小分子三者相互作用的结合信号。目前最新报道的aTF-NAST(aTF-based nicked DNA-template-assiated signal transduction)等系统进一步提高了尿酸和对羟基苯甲酸的灵敏度，但是样品分析周期长，成本高，难以实现快速，批量检测。因此，目前本领域仍然急需建立一种简单、快速、灵敏度高、特异性强以及适合于高通量体外检测小分子化合物的方法。
CRISPR/Cas系统是细菌及古细菌在RNA引导下利用Cas蛋白抵御外源核酸入侵的过程中演化而来的一种获得性免疫防御机制。1978年首次报道了一种功能未知的成簇的重复DNA序列，直至2001年正式命名为成簇规律间隔短回文重复序列(Clustered Regularly Interspersed Palindromic Repeats，CRISPR)。随着近年来科研工作者的不懈探索，对CRISPR系统的分类和作用机制等得到初步解析并且已广泛应用于基因编辑、DNA组装以及基因表达调控等各个领域。
根据Cas(CRISPR-associated proteins，CRISPR相关蛋白)基因的组成和效应蛋白的数量，CRISPR/Cas系统被分为了2类5型；1类CRISPR/Cas系统利用多个效应蛋白复合物干扰靶基因；2类CRISPR/Cas系统利用单一的效应蛋白抵御外源核酸的入侵，因其简单高效受到广泛关注。其中，Cas9和Cas12a(也称为Cpf1)作为2类CRISPR/Cas系统的典型，已成功应用于原核/真核细胞基因编辑和基因调控领域。近期基于Cas12a的反式切割活性，Doudna团队开发的“DETECTR”(DNA endonuclease targeted CRISPR trans reporter)以及赵国屏团队表征的“HOLMES”(an one-HOur Low-cost Multipurpose highly Efficient System)的诊断系统，进一步推进了CRISPR/Cas12a系统在核酸检测领域的应用前景。但是目前仍未有将CRISPR/Cas系统用于检测小分子化合物的相关报道。
发明内容
在第一方面，本发明提供了一种用于检测小分子的生物传感器，所述生物传感器包含识别元件和换能元件，其中，
A：所述识别元件包含别构转录因子(aTF)和激活双链DNA(激活dsDNA)，所述激活dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点(即，原型间隔区邻近基序，PAM)以及与向导RNA至少部分互补的序列，以及
所述换能元件包含所述CRISPR/Cas12a系统以及单链DNA探针(ssDNA探针)，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白和所述向导RNA(gRNA)，所述ssDNA探针两端分别缀合有发光/生色基团及其淬灭基团；
或者
B：所述识别元件包含别构转录因子(aTF)和双链DNA探针(dsDNA探针)，所述dsDNA探针包含所述别构转录因子的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA(gRNA)至少部分互补的序列，并且所述dsDNA探针两端分别缀合有发光/生色基团及其淬灭基团，以及
所述换能元件包含所述CRISPR/Cas12a系统，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述gRNA。
在本发明的实施方式中，当所述生物传感器与目标小分子接触后，所述aTF与目标小分子的结合使得所述别构转录因子与所述激活dsDNA和/或dsDNA探针的结合亲和力升高或降低，从而使游离形式的激活dsDNA和/或dsDNA探针减少或增加；所述游离形式的激活dsDNA作为激活剂与CRISPR/Cas12a蛋白和所述gRNA形成三元复合物，从而激活CRISPR/Cas12a蛋白的反式ssDNA切割活性，使得ssDNA探针被切割并产生可检测的光信号，或者，所述游离形式的dsDNA探针被所述gRNA识别并被CRISPR/Cas12a蛋白通过顺式切割活性进行切割，从而产生可检测的光信号；通过所产生的光信号，可以对所述目标小分析的存在和/或含量进行检测。
在第二方面，本发明提供了一种用于检测小分子的试剂盒，所述试剂盒包含第一方面所述的生物传感器。在一些实施方式中，所述试剂盒还包含用于对光信号进行检测的检测元件。
在第三方面，本发明提供了一种对样品中的小分子进行检测的方法，所述方法包括：将所述样品与第一方面所述的生物传感器接触，以生成光信号，以及对所产生的光信号进行检测，从而检测所述样品中的小分子。
在第四方面，本发明提供了一种用于对小分子进行检测的组合物，所述组合物包含识别试剂和换能试剂，其中，
A：所述识别试剂包含别构转录因子(aTF)和激活双链DNA(激活dsDNA)，所述激活dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列，以及
所述换能试剂包含所述CRISPR/Cas12a系统和单链DNA探针(ssDNA探针)，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)，所述ssDNA探针两端分别缀合有发光/生色基团及其淬灭基团；
或者
B：所述识别试剂包含aTF和双链DNA探针(dsDNA探针)，所述dsDNA探针包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA(gRNA)至少部分互补的序列，并且所述dsDNA探针两端分别缀合有发光/生色基团及其淬灭基团，以及
所述换能试剂包含CRISPR/Cas12a系统，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述gRNA。
在第五方面，本发明提供了一种用于检测小分子的试剂盒，所述试剂盒包含第五方面所述的组合物。
在第六方面，本发明提供了一种用于对小分子进行检测的方法，所述方法包括使用第五方面所述的组合物或第六方面的试剂盒与待测样品接触，以生成光信号，以及对所产生的光信号进行检测，从而检测所述小分子。
第七方面，本发明提供了第一方面所述的生物传感器、第四方面所述的组合物和第二方面或第五方面所述的试剂盒在检测小分子方面的用途。
有益效果
相比传统的小分子检测方法，本发明采用aTF与CRISPR/Cas12a介导的小分子检测工具
( CRISPR/Cas12a-and  aTF-mediated  small  mol ecu le detect or，简称为“CaT-SMelor”)，通过目标小分子与aTF的结合使得aTF与双链DNA的亲和力变化以及CRISPR/Cas12a的顺式切割活性和反式单链DNA切割活性，对dsDNA探针和/或ssDNA探针进行切割，将目标小分子信号转变为相应的光信号，而不涉及aTF-靶DNA结合的DNA转录调控过程。本发明所述的检测方法不仅操作简便、节约时间(至多25分钟，甚至只需要5-10分钟)和节约成本(每次反应只需不到2元)，而且灵敏度高(检测极限降至nM级)、准确度高以及反应体系小(例如，血液样品仅需1μL)，可于96孔板或384孔板上进行操作，因此适合于诊断目的和非诊断目的的高通量精确检测。
附图说明
图1为根据本发明的一个实施方式，利用本发明的生物传感器进行小分子检测的原理示意图。在本实施方式中，为了便于后续操作和提高检测灵敏度，对aTF进行了固定化。具体而言，将aTF与纤维素结构域(CBD)融合表达，然后将融合蛋白aTF-CBD与微晶纤维素(MC)混合使其固定在MC上；融合蛋白aTF-CBD与激活dsDNA结合形成aTF-CBD-DNA复合物；小分子与aTF的结合导致aTF变构效应，使得激活dsDNA从aTF上脱离，得到游离形式的激活dsDNA(也称为“游离dsDNA”)；将游离形式的激活dsDNA加入CRISPR/Cas12a系统和ssDNA探针(报告子：FQ标记的ssDNA)中，游离的激活dsDNA激活CRISPR/Cas12a系统并使其反式切割探针，使得发出光信号(荧光信号)；然后对光信号进行检测。根据本发明的实施方式，使用的血液样品需求量较少(～1μL)，而且反应体系体积少，可在96/384孔板上进行操作。使用酶标仪等常用仪器检测光信号，从而实现对小分子的定性/定量分析。
图2为说明根据本发明的实施方式，通过凝胶阻滞实验(EMSA)对实施例1-实施例2中制备的融合蛋白CBD-aTF(CBD-HucR、CBD-HosA和CBD-TetR)与dsDNA的结合能力，以及在存在特定小分子的情况下CBD-aTF-dsDNA的解离的图。图2A：融合蛋白CBD-HosA、CBD-HucR和CBD-TetR的结构示意图。别构转录因子HucR、HosA和TetR与CBD之间通过接头连接(所述接头的氨基酸序列可如SEQ ID NO：12所示)；为了便于纯化和识别，融合蛋白上还具有His标签。图2B-图2C：示出了在不存在或存在尿酸的情况下，融合蛋白CBD-HucR与激活dsDNA(dsDNA (HucR))的结合的图(采用EMSA测量)。图2D-图2E：示出了在不存在或存在四环素的情况下，融合蛋白CBD-TetR与激活dsDNA(dsDNA (TetR))的结合的图(采用EMSA测量)。图2F-图2G：示出了在不存在或存在对羟基苯甲酸(p-HBA)的情况下，融合蛋白CBD-HosA与激活dsDNA(dsDNA (HosA))的结合的图(采用EMSA测量)。
图3为示出了根据本发明的一个实施方式，使用本发明实施例1-实施例2的生物传感器检测靶小分子的图表。图3A：示出了使用本发明实施例1-实施例2的生物传感器检测小分子的流程图。图3B：示出了在存在500μM尿酸及其结构类似物(腺嘌呤、鸟嘌呤、次黄嘌呤)的情况下，融合蛋白CBD-HucR与激活dsDNA(dsDNA (HucR))的结合的图(采用EMSA测量)。图3C：示出了以500μM尿酸(UA)及其结构类似物(腺嘌呤、鸟嘌呤、次黄嘌呤)作为样品，使用本发明实施例1的尿酸生物传感器所检测到的荧光强度的图表，其中，以40nM游离的激活dsDNA(dsDNA (HucR))作为阳性对照(PC)。图3D：示出了在存在1.8mM对羟基苯甲酸(p-HBA)及其结构类似物(酪醇(Tyrosol，也称为“对羟苯基乙醇”)、对氨基苯甲酸(p-ABA)、对羟基苯甲酸甲酯(p-MHB)、对羟基苯甲醇(p-HBnOH))的情况下，融合蛋白CBD-HosA与激活dsDNA(dsDNA (HosA))的结合的图(采用EMSA测量)。图3E：示出了以1.8mM p-HBA及其结构类似物(Tyrosol、p-ABA、p-MHB、p-HBnOH)作为样品，使用本发明实施例2的p-HBA生物传感器所检测到的荧光强度的图表，其中，以40nM游离的激活dsDNA(dsDNA (HosA))作为阳性对照(PC)。
图4为示出了根据本发明的实施方式，使用本发明实施例1-实施例2的生物传感器对靶小分子进行检测的图表。图4A：不同浓度的激活dsDNA对荧光强度变化的影响的检测结果，并且取荧光值线性增加区间的斜率，以激活dsDNA浓度为横坐标，对应的斜率为纵坐标计算出表示激活dsDNA线性检测范围的回归方程。图4B：不同浓度的尿酸对荧光强度变化的影响的检测结果，并且取荧光值线性增加区间的斜率， 以尿酸浓度为横坐标，对应的斜率为纵坐标计算出尿酸的线性检测范围为25-500nM。图4C：不同浓度的p-HBA对荧光强度变化的影响的检测结果，并且取荧光值线性增加区间的斜率，以p-HBA浓度为横坐标，对应的斜率为纵坐标计算出p-HBA的线性检测范围为9-180nM。
图5为示出了根据本发明的实施方式，与传统尿酸浓度测量方法(HPLC以及Backman试剂盒)相比，使用本发明实施例1的尿酸生物传感器对人血液样品中的尿酸浓度进行测定的图表。图5A：示出了使用本发明的尿酸生物传感器与HPLC方法检测人血液样品的尿酸的图表。图5B：示出了使用本发明的尿酸生物传感器与临床上使用的Backman试剂盒检测人血液样品中的尿酸的图表。图5C：示例性地说明了在血液样品分析中使用本发明的生物传感器的流程图。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
本发明利用本发明采用CaT-SMelor，通过目标小分子与aTF的结合使得aTF与双链DNA的亲和力变化以及CRISPR/Cas12a的顺式切割活性和反式单链DNA切割活性，对dsDNA探针和/或ssDNA探针进行切割，将目标小分子信号转变为相应的光信号，实现对小分子的定性检测。进一步，本发明人发现，对于目标小分析而言，在一定浓度范围内，所产生的荧光曲线斜率的变化与小分子的浓度成正比，因此通过对含有目标小分子的样品的荧光曲线斜率进行测量可实现对样品中目标小分子的精确定量检测。
组合物
本发明提供了一种用于对小分子进行检测的组合物，所述组合物包含识别试剂和换能试剂，其中，
所述识别试剂包含别构转录因子(aTF)和双链DNA(dsDNA)，所述dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列；以及所述换能试剂包含所述CRISPR/Cas12a系统，
其中，所述组合物中还含有发光/生色基团。
在一个实施方式中，所述换能试剂进一步包含单链DNA(ssDNA)。
具体而言，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)。
在本发明的具体实施方式中，所述dsDNA可作为激活dsDNA，或dsDNA探针。在另一实施方式中，所述ssDNA也可作为探针使用。在作为探针使用的情况中，所述dsDNA探针或ssDNA探针的两端可缀合有发光/生色基团及其淬灭基团。
在一个实施方式中，本发明的组合物包含识别试剂和换能试剂，所述识别试剂包含别构转录因子(aTF)和激活双链DNA(激活dsDNA)，所述激活dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列，以及
所述换能试剂包含所述CRISPR/Cas12a系统和单链DNA探针(ssDNA探针)，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)，所述ssDNA探针两端分别缀合有发光/生色基团及其淬灭基团。
在另一实施方式中，本发明的组合物包含识别试剂和换能试剂，所述识别试剂包含aTF和双链DNA探针(dsDNA探针)，所述dsDNA探针包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA(gRNA)至少部分互补的序列，并且所述dsDNA探针两端分别缀合有发光/生色基团及其淬灭基团，以及
所述换能试剂包含CRISPR/Cas12a系统，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述gRNA。
在本发明的实施方式中，本发明的组合物可处于生物传感器的形式。
生物传感器
生物传感器(biosenser)通常被认为是对生物材料(如组织、微生物细胞、细胞器、细胞受体、酶、抗体、核酸等)或生物衍生材料或生物模拟材料进行分析的一类器件，它将生物材料等与物理化学传感器或传感微系统(可以是光学的、电化学的、热学的、压电的或磁学的)密切结合或联系起来，能够产生间断或连续的信号(光信号、电信号等)，信号强度与被分析物成比例(例如线性关系)，从而行使分析功能。在本领域中，生物传感器具有通用的定义，通常由两部分构成：识别元件(又称为分子识别元件)和换能元件(又称为换能器)。在本文中，术语“生物传感器”即指包含识别元件和换能元件的器件或设备，也称为“生物传感平台(biosening platform)”。不受理论地束缚，本文所述的生物传感器可包括进一步提高检测灵敏度的放大元件和/或直接进行检测的检测元件。此外，本文所述的生物传感器并不限于具体的形式，只要包含下面描述的识别元件和换能元件的任何形式均包含在本文术语“生物传感器”的范围内。
识别试剂或识别元件
识别元件是生物传感器的关键元件，其识别待分析物，发生生物学反应，直接决定传感器的功能与质量。在本文中，所述识别元件包含aTF和激活dsDNA，或者aTF和dsDNA探针。
本发明所使用的术语“别构转录因子”具有本领域公知的含义。为适应外界环境的改变并及时做出应答，微生物进化出别构蛋白aTF作为感知环境的重要输入元件，aTF通常包含一个DNA结合结构域(DBD)和效应物结合结构域(EBD)，其中效应物的结合改变aTF与靶基因序列的结合，从而调控相关基因的表达，快速响应外界的变化。根据aTF序列相似性以及结构和功能的不同，可分为不同家族如AraC、LacI、TetR等，这些aTF往往能够特异性感知各类小分子化合物，对于维持新陈代谢的稳定发挥重要作用。
别构转录因子包含与效应物(通常为小分子)结合的结构域(EBD)以及DNA结合结构域(DBD)，其与效应物的结合引起构象改变，使得该别构转录因子和与其特异性相互作用的DNA片段(天然状态下通常为启动子操纵序列)的结合亲和力发生改变，从而增强或减弱由该操纵序列控制的DNA序列的转录(Nat Methods.2016，13(2)：177-183)，通过这样的方式使得基因的转录依赖于小分子的浓度。在原核生物中，操纵序列通常出现在与代谢有关的操纵子或报告基因的上游；别构转录因子常常起到针对细胞内效应物小分子的传感器的作用，反馈小分子的浓度信息，从而对细胞内的生物合成途径进行动态调控。在真核生物中，此类别构转录因子常存在于控制细胞分化与个体发育的通路中。鉴于本发明中别构转录因子与DNA的相互作用发生在体外，原核和真核来源的别构转录因子均可作为本发明的识别元件。在本发明中，转录因子结合位点也称为转录因子作用位点、转录因子操作位点或转录因子结合基序。不希望被理论所限地，转录因子作用位点通常为互补的双链DNA。此外，本领域技术人员应当知晓，转录因子作用位点附近存在单个3',5'-磷酸二酯键断裂(缺口)不影响别构转录因子与其作用位点的结合。
天然转录系统中转录因子作用位点的序列是已知的。不同物种的转录因子结合位点长度略有不同。转录因子结合位点在大肠杆菌中的平均长度为24.5bp，在果蝇中为12.5bp(J Mol Biol.1998，284(2)：241-54；Nucleic Acids Res.2003，31(1)：374-8)。在本发明中，转录因子作用位点的长度优选10-40bp、更优选15-25bp、最优选17-19bp。与别构转录因子作用的DNA片段并不限于在天然系统中存在的别构转录因子作用位点。通过对作用位点序列进行随机或定向突变，或者结合计算机模拟选出数条候选序列并验证别构转录因子与各DNA片段的结合能力是本领域成熟的技术(Mol Microbiol.2005，55(3)：712-23.)。另外，也可对别构转录因子的小分子结合结构域和/或DNA结合结构域进行突变，优化别构转录因子与小分子和/或DNA的结合亲和力。可通过定向突变和进化，提高别构转录因子和与该别构转录因子作用的DNA片段之间的平衡解离常数和/或降低别构转录因子与小分子之间的平衡解离常数，进一步提高本发明传感器的灵敏度。
如上所述，别构转录因子与小分子的结合可使得所述别构转录因子与靶dsDNA片段(即包含别构转录因子作用位点的DNA片段，在本文中也称为“激活dsDNA”或“dsDNA探针”)的结合亲和力升高(激活系统)或降低(阻遏系统)。在一些实施方式中，本发明的别构转录因子为激活系统的别构转录因子。在该系统中，别构转录因子与效应物小分子的结合使得其与靶dsDNA的结合亲和力增强(即，小分子的结合使得别构转录因子能够结合于靶dsDNA片段)，从而使游离的靶dsDNA(即未与别构转录因子结合的靶dsDNA)的量减少。优选地，所述别构转录因子与所述小分子结合后形成的转录因子-小分子复合体与所述靶dsDNA片段结合的平衡解离常数大于所述别构转录因子与所述靶dsDNA片段结合的平衡解离常 数；优选地，所述别构转录因子-小分子复合体与靶dsDNA片段结合的平衡解离常数是所述别构转录因子与所述靶dsDNA片段结合的平衡解离常数的10-10000倍、优选20-5000倍、更优选50-1000倍。在一些实施方式中，本发明的别构转录因子为阻遏系统的别构转录因子。在该系统中，别构转录因子与效应物小分子的结合使得其与靶dsDNA的结合亲和力减弱(即，小分子的结合使得别构转录因子从其作用位点处脱离，游离的靶dsDNA的量增加)。优选地，所述别构转录因子与所述小分子结合的平衡解离常数大于所述别构转录因子与所述靶dsDNA片段结合的平衡解离常数；优选地，所述别构转录因子与所述小分子结合的平衡解离常数是所述别构转录因子与本发明的别构转录因子为所述靶dsDNA片段结合的平衡解离常数的10-10000倍、优选20-5000倍、更优选50-1000倍。在一些实施方式中，所述别构转录因子和靶dsDNA作为组合物或结合成复合体提供。在另一些实施方式中，所述别构转录因子和靶dsDNA作为单独的化合物提供。应当特别指出的是，虽然在本发明的具体实施例中仅采用阻遏系统的别构转录因子，但是其仅仅是说明性的，而基于本申请的教导，本领域技术人员可根据实际需要采用不同的别构转录因子(例如其它的阻遏系统的别构转录因子或者激活系统的别构转录因子)来实施本发明。
本发明的生物传感器、组合物、试剂盒和方法可用于诊断用途或非诊断用途、或者非临床用途，例如用于环境污染监控、食品和化妆品质量控制和疾病诊断。在本发明中，作为检测对象的小分子为使得别构转录因子构象变化的效应物。一般而言，小分子的特征在于它具有大于约50道尔顿但小于约5000道尔顿(5kD)的分子量。优选小分子具有小于1kD的分子量。在本发明中，小分子可为例如环境指标、疾病指标或健康指标，包括但不限于重金属离子、毒素、药物、代谢物、污染物或上述物质的分解产物等。小分子可存在于环境中或为细菌、真菌、植物或动物源性，或者为人工合成。甚至对于不存在相对应的别构转录因子的效应物小分子，本领域技术人员能够通过计算机模拟设计将效应物结合结构域添加至DNA结合结构域，或改造天然转录因子的效应物结合结构域，构建人工的别构转录因子(Nat Methods.2016，13(2)：177–183)。
在本发明中，待检测的小分子可存在于任何液体样品或可通过适当操作转换为液体样品的固体样品中。样品可为环境样品，例如为地下水、中水、海水、废水、采矿废料的样品。或者，样品可为生物样品，特别是来自受试者的样品，例如以下样品中的一种或多种：血液、血清、血浆、痰、脑脊液流体、尿液、泪液、肺泡分离物、胸膜液、囊液、组织、唾液。样品也可来自于食品、饮用水、化妆品或饲料。
在一些实施方式中，可对样品进行预处理，从而对待检测的小分子进行富集和提取，或者去除可能干扰检测的杂质。例如，可通过离心、过滤、超声、匀浆化、加热、冷冻、解冻、机械处理或多种操作方法的组合，和/或加入预处理的试剂。本领域技术人员知晓针对特定样品的常见预处理方法和预处理试剂。例如，常用的预处理试剂包括表面活性剂和洗涤剂、盐、细胞裂解剂、抗凝血剂、降解酶(例如蛋白酶、脂酶、核酸酶、脂肪酶、胶原酶、纤维素酶、淀粉酶等)以及溶液(例如缓冲液)等。
表1列出了多种别构转录因子、与其相互作用的DNA序列及对应的效应物小分子。本领域技术人员能够理解的是，用于本发明的别构转录因子并不限于所列举的这些。另外，也可对与所列举的别构转录因子相互作用的DNA序列的一个或多个碱基进行替换、删除或加入一个或多个碱基，从而改变别构转录因子和与其相互作用的DNA序列的结合强度。
表1：示例性的别构转录因子、与其相互作用的DNA序列以及所对应的效应物
Figure PCTCN2020084111-appb-000001
Figure PCTCN2020084111-appb-000002
在本发明的一些实施方式中，所述识别元件包含可固定在介质上的别构转录因子和/或可固定在介质上的激活dsDNA或dsDNA探针。在一些实施方式中，所述识别元件包含可固定在介质上的别构转录因子。在一些实施方式中，所述识别元件包含可固定在介质上的激活dsDNA或dsDNA探针。在一些实施方式中，所述识别元件包含可固定在介质上的别构转录因子和可固定在介质上的激活dsDNA或dsDNA探针。术语“介质”是指支撑各个元件并为各个元件提供反应空间从而便于携带、运输、包装或操作的物质。本领域技术人员通常通过固定化技术将需要固定的元件固定在介质上，例如吸附法、共价键合法、物理包埋法和交联法等本领域通常使用的方法。介质的实例包括但不限于硝化纤维素膜或尼龙膜、亲和柱层析基质、磁珠、固体填料、微晶纤维素、或可商购的蛋白质固定化介质等。在优选的实施方式中，所述介质为微晶纤 维素。在一些优选的实施方式中，本发明所述的识别元件包含可固定在微晶纤维素上的别构转录因子。在进一步优选的实施方式中，所述可固定在微晶纤维素上的别构转录因子是与纤维素结构域(以下简称“CBD”)融合表达的别构转录因子，该融合蛋白通过CBD与微晶纤维素之间的吸附作用而可固定在微晶纤维素上。
在本发明的一些实施方式中，所述识别元件包含与CBD融合蛋白表达的别构转录因子。如具体实施例所示的，本发明人将纤维素结构域与别构转录因子进行融合表达，从而获得可固定在微晶纤维素上的融合蛋白。例如，此类融合蛋白的结构示意图如图2A所示，将别构转录因子(如HucR、HosA、TetR，其氨基酸序列可分别如SEQ ID NOs:1-3所示)与CBD(其氨基酸序列可如SEQ ID NO:4所示)以接头连接。所述接头的目的仅仅是将aTF与CBD连接起来，其氨基酸序列可如图2A所示(SEQ ID NO:12)，但并不限于此，本领域技术人员可根据实际需要选择其它接头。本发明人通过实验证实了，结构转录因子以融合蛋白形式表达(CBD-HucR、CBD-HosA和CBD-TetR)并没有不利地影响其与激活dsDNA的结合能力以及变构活性(如图2B-图2G所示)。并且，比较融合蛋白和单独表达的别构转录因子之间的DNA结合能力和变构活性，发现两者之间也没有明显区别。这说明CBD仅仅起到将别构转录因子固定至微晶纤维素的目的，而并不对别构转录因子的功能产生影响。将别构转录因子与CBD融合表达并纯化的方法是本领域熟知的。例如，将编码氨基酸序列如SEQ ID NOs:1-3所示的别构转录因子的基因和编码氨基酸序列如SEQ ID NO:4所示的纤维素结构域的基因克隆至表达载体(例如含有His-标签的pET23b载体)上，构建pET23b-aTF-CBD质粒；将该质粒转入表达菌(大肠杆菌BL21)，诱导表达即可获得融合蛋白。进一步，可采用亲和柱层析、快速蛋白液相色谱等本领域通常使用的蛋白质纯化方法进行纯化。在本发明中，融合蛋白包含经纯化的融合蛋白。不受具体理论的束缚，也可将本发明的其它元件(例如下文所述的换能元件或检测元件)固定在介质上，本领域技术人员可根据实际需要进行修改或改进，这些改进或修改也包含在本发明的范围内。
本发明所使用的术语“激活dsDNA”或“激活dsDNA片段”为包含别构转录因子作用位点、CRISPR/Cas12a蛋白识别位点PAM的DNA片段和与gRNA至少部分互补的序列的核苷酸，其至少在别构转录因子作用位点、CRISPR/Cas12a蛋白识别位点和与gRNA至少部分互补的序列处为双链。所述激活dsDNA可作为激活剂激活CRISPR/Cas12a系统的反式切割活性，因此在本文中将其称为“激活dsDNA”，也因其介导换能元件启动，也可被称为“换能DNA”。在本发明中，所述激活dsDNA的长度优选为20-80bp、更优选55-65bp，最优选58-60bp。其中，关于别构转录因子结合位点的定义如上所述。
本文所述的CRISPR/Cas12a蛋白识别位点PAM是指可被CRISPR/Cas12a系统识别并因此激活CRISPR/Cas12a系统的切割活性的序列，该序列称为原型间隔区邻近基序(PAM)，一般富含T(对于CRISPR/Cas12a)。
在本发明中，PAM的长度优选3-8bp、更优选3-6bp、最优选4bp。PAM序列并不限于在天然系统中存在的PAM。通过对作用位点序列进行随机或定向突变，或者结合计算机模拟选出数条候选序列并验证PAM与CRISPR/Cas12a的结合能力是本领域已知的技术。例如，在本发明的实施方式中，所述激活dsDNA中的别构转录因子作用位点的核苷酸序列以及PAM序列如下所示(SEQ ID NO:5-7)：
tactgagccatgtatccaggtca ttgTACTTAGATGTCTACCTAagctctgacagttcca、
tactgagccatgtatccaggtca ttgCGTTCGTATACGAACAgtagctctgacagttcca、
tgagccatgtatccaggtca tttgTCCCTATCAGTGATAGAGAagctctgacagttcca(其中，下划线部分为PAM，黑体部分为别构转录因子结合位点，所述别构转录因子结合位点即为与gRNA互补的部分)。
在本发明中，所述与gRNA“至少部分互补”是指与所述gRNA至少50％以上互补，例如至少70％以上互补，例如80％、85％、90％、95％、99％以上或100％互补的序列。
在本发明的实施方式中，在激活dsDNA中，所述与gRNA至少部分互补的序列可在转录因子结合位点和PAM之前、之后或者之间，或者与转录因子结合位点和/或PAM至少部分重叠，或者位于别构转录因子结合位点之内。例如，在激活dsDNA中，别构转录因子结合位点即为所述与gRNA至少部分互补的序列。因此，在一个优选的实施方式中，激活dsDNA中可仅包含PAM和别构转录因子结合位点(其中，含有与gRNA互补的序列)。因此，激活dsDNA的长度可低至13或14bp。
在本发明中，在“激活dsDNA”中，所述别构转录因子作用位点与PAM序列之间可间隔数个核苷酸，例如可间隔0-20bp、更优选5-10bp、最优选6-9bp。在一个实施方式中，所述别构转录因子作用位点与PAM序列之间可直接连接，或者可部分或完全重叠。或者，PAM序列可位于别构转录因子作用位点中。
在本文中，所述“激活dsDNA”上同时存在别构转录因子作用位点和CRISPR/Cas12a蛋白识别位点PAM，因而可以同时与别构转录因子和Cas12a蛋白结合。所述别构转录因子与所述激活dsDNA片段结合的平衡解离常数可高于、低于或相似于所述与CRISPR/Cas12a蛋白与所述激活dsDNA片段结合的平衡解离常数。在本发明的一些实施方式中，所述别构转录因子与所述激活dsDNA片段结合的平衡解离常数低于所述CRISPR/Cas12a蛋白与所述激活dsDNA片段结合的平衡解离常数，即相比于别构转录因子，所述激活DNA片段更容易结合至CRISPR/Cas12a蛋白。因此为了减少别构转录因子和CRISPR/Cas12a系统对彼此功能的干扰，降低噪音、提高灵敏度，本发明的别构转录因子和CRISPR/Cas系统可以单独的形式提供并且在不同的空间进行反应。在一些实施方式中，所述激活dsDNA可通过如下方法制备：例如采用靶向特定别构转录因子作用位点和CRISPR/Cas蛋白识别位点的引物对(例如，核苷酸序列如SEQ ID NOs:5-18、6-19或7-20所示)通过退火(例如95℃预变性5min；95℃变性30s，每个循环降低1℃，70个循环；25℃保存)形成双链DNA；或者也可通过人工合成，例如人工合成核苷酸序列如SEQ ID NOs:5-7所示的双链DNA。所述激活dsDNA也可通过本领域已知的其它方式进行制备。
在一个实施方式中，为了避免在相同反应体系中aTF与CRISPR/Cas12a蛋白竞争性结合激活dsDNA，可通过将aTF与不影响其活性的其它蛋白(例如，CBD)融合表达，或者使得激活dsDNA中别构转录因子作用位点与PAM序列存在重叠。
在本文中可互换使用的“探针”或“DNA探针”是指与gRNA互补，并被所述CRISPR/Cas12a系统切割并且具有在被切割后可产生可检测光信号的基团(在本文中也称为“标记”)的核苷酸序列。在一些实施方式中，所述探针具有双链区域，或者为双链DNA探针(均称为dsDNA探针)，所述双链DNA区域含有别构转录因子作用位点、CRISPR/Cas12a系统的识别位点PAM和可被gRNA和/或crRNA识别的特定核苷酸序列，即与gRNA和/或crRNA至少互补的序列。在一个实施方式中，所述dsDNA探针的长度例如为13-100bp、更优选20-50bp、最优选20-30bp。其中，关于别构转录因子结合位点的定义如上所述。在具体的实施方式中，dsDNA探针的核苷酸序列部分可与所述激活dsDNA相同。在本发明中，可被gRNA和/或crRNA识别的序列长度优选15-70bp、更优选15-30bp、最优选17-24bp。在优选的实施方式中，dsDNA探针包含与gRNA和/或crRNA至少70％以上互补，例如80％、85％、90％、95％、99％以上或100％互补的序列。
在本发明中，在“dsDNA探针”中，所述别构转录因子作用位点与可被gRNA和/或crRNA识别的序列之间可间隔数个核苷酸，例如可间隔0-20bp、更优选5-10bp、最优选6-9bp。在一个实施方式中，所述别构转录因子作用位点与可被gRNA和/或crRNA识别的序列之间可直接连接，或者可部分或完全重叠。或者，可被gRNA和/或crRNA识别的序列可位于别构转录因子作用位点中。
所述在一些实施方式中，所述探针是单链DNA，所述单链DNA可以为任意核苷酸序列，例如长度为约10-30bp、优选约20bp的核苷酸序列，如SEQ ID NO:8所示的核苷酸序列。不受具体理论的限制，在本发明的激活DNA是双链DNA且可设计成被gRNA和/或crRNA所识别的情况下，根据本发明的意图和原理，本领域技术人员可将本发明的激活dsDNA设计成双链DNA探针(例如在激活DNA两端进行荧光标记)来实施本发明，这一修改也应包括在本发明的范围内。
换能试剂或换能元件
在本文中，所述“CRISPR/Cas系统”是指2类CRISPR/Cas系统，其利用单一的效应蛋白抵御外源核酸的入侵，能够简单高效进行基因编辑和核酸检测。其中，最典型的2类CRISPR/Cas系统是CRISPR/Cas12a(Cpf1)系统。在本文中，CRISPR/Cas系统通常至少包含CRISPR/Cas蛋白和向导RNA。CRISPR(成簇规律间隔短回文重复)是含有短碱基序列重复的原核DNA片段。每个重复之后是来自以前暴露于细菌病毒或质粒的“间隔子DNA”的短片段。Cas(CRISPR相关蛋白)是一种RNA(向导RNA)引导的DNA核酸内切酶。向导RNA(gRNA或sgRNA)是引导CRISPR/Cas蛋白识别和切割目标核酸分子的特定RNA序列，可通过体外转录或者人工化学合成。向导RNA可由CRISPR RNA(crRNA)和反式激活crRNA (tracrRNA)杂交形成，或者可作为单独的连续RNA提供。gRNA经由crRNA部分中的靶特异性序列(例如，间隔序列“间隔子”)特异性结合至互补靶序列，而CRISPR/Cas蛋白自身结合至PAM，然后Cas核酸酶介导靶核酸(例如DNA探针)的切割。然而，对于CRISPR/Cas12a，仅需要crRNA引导即可，而不需要tracrRNA；并且CRISPR/Cas12a不仅对特定序列的DNA双链(dsDNA，其含有与crRNA互补的序列被crRNA所识别)进行切割而且在被特定序列的DNA双链(如本文的激活DNA)激活形成CRISPR/Cas/dsDNA三元复合物时可对任意的单链DNA(ssDNA)进行切割。这些特性使得CRISPR/Cas12a能够提高检测的灵敏性、特异性和速度。因此，在一些实施方式中，CRISPR/Cas12a系统是优选的。就本发明人所知，Cas12a自身并不能用于在疾病诊断、药物研发、食品安全以及环境监测等各个领域对小分子化合物进行检测。而在本发明中，本发明人借助识别元件(别构转录因子-激活dsDNA复合体)，当激活dsDNA上的PAM结合CRISPR/Cas蛋白激活CRISPR/Cas对探针(靶核酸)进行切割，从而产生可检测的光信号，得以实现对小分子化合物的检测。
在本发明中，gRNA的长度优选20-70bp、更优选30-50bp、最优选38-45bp。gRNA可基于使用者的需求进行设计。优选结合生物信息学软件来进行设计。在一些实施方式中，所述光信号包括荧光信号或吸收光信号。本领域技术人员应当理解，为了量化在分析中出现的颜色变化，通常采用特定波长的光进行激发后检测吸收光强度。因此，在本发明中，术语“吸收光信号”也可指产生的颜色变化(比色分析)。在一些实施方式中，所述探针可携带标记从而在被CRISPR/Cas切割后产生可检测的光信号。标记的实例包括但不限于：发光有机化合物(如荧光素、萝卜素)、发光无机化合物(例如，化学染料)、荧光团(如FAM荧光团)等；纳米颗粒和量子点等；或者生色团等。利用上述物质对核酸序列进行标记使其产生可检测的光信号的技术是本领域技术人员熟知的，并且可根据实际需要进行选择和改进，这一点并不会对本发明进行限制。在一些优选的实施方式中，所述光信号为荧光信号。在进一步优选的实施方式中，所述标记为发光团和淬灭团，分别标记在探针的两端(3’端或5’端)。优选地，所述发光团为FAM荧光团，所述淬灭团为BHQ荧光淬灭团。在另一些优选的实施方式中，所述光信号为吸收光信号。在进一步优选的实施方式中，所述标记为生色团和淬灭团，分别标记在探针的两端(3’端或5’端)。
检测元件
在本发明中，利用各种检测设备对上述光信号进行检测是本领域技术人员所熟知的。
检测方法
在本发明的实施方式中，使用本发明所述的生物感受器或本发明所述的组合物对样品中的小分子进行检测。在本发明中，可对样品中的小分子进行定性或定量检测。
本发明提供了一种对待测样品中的小分子进行检测的方法，所述方法包括：
将待测样品与识别试剂进行混合并孵育；从所得的混合物中分离游离的dsDNA片段；将分离的游离dsDNA加入换能试剂中，并检测所产生的光信号；以及，基于所产生的信号来检测所述小分子的存在或含量。
在一个实施方式中，本发明提供了一种对待测样品中的小分子进行检测的方法，所述方法包括：
(1)将待测样品、aTF和dsDNA进行混合并孵育；(2)从所得的混合物中分离游离的dsDNA片段；(3)将分离的游离dsDNA加入CRISPR/Cas12a蛋白和向导RNA中，并检测所产生的光信号；以及(4)基于所产生的信号来检测所述小分子的存在或含量。
在一个方面，本发明提供了一种对待测样品中的小分子进行检测的方法，所述方法包括：
(1)将待测样品、aTF和激活dsDNA片段进行混合并孵育；
(2)从步骤(1)中的混合物分离游离的激活dsDNA片段；
(3)向步骤(2)中分离的游离激活DNA片段中加入CRISPR/Cas12a蛋白、向导RNA和ssDNA探针，并检测所产生的光信号；以及
(4)基于所产生的信号，对所述小分子的存在或含量进行检测。
在另一方面，本发明提供了一种对待测样品中的小分子进行检测的方法，所述方法包括：
(1)将待测样品、aTF和dsDNA探针进行混合并孵育；
(2)从步骤(1)中的混合物分离游离的dsDNA探针；
(3)向步骤(2)中分离的游离dsDNA探针中加入CRISPR/Cas12a蛋白和向导RNA，并检测所产生的光信号；以及
(4)基于所产生的信号，对所述小分子的存在或含量进行检测。
其中，步骤(1)中进行混合和孵育的具体条件可由本领域技术人员根据实际需要选择，只要保证别构转录因子和激活dsDNA或dsDNA探针能充分结合成复合体并且待测样品中的特定小分子化合物可以与别构转录因子-激活dsDNA或dsDNA探针复合物充分结合即可，例如在室温下反应1min以上，例如15-20min(但不限于此)。对于别构转录因子和激活dsDNA或dsDNA探针的比例，根据实际需求可以进行调整。在本发明的一些实施方式中，别构转录因子和激活dsDNA或dsDNA探针的摩尔比≥5:1是优选的，此时，别构转录因子可与激活dsDNA或dsDNA探针完全结合形成蛋白-DNA复合物。
在一些实施方式中，所述别构转录因子为阻遏系统的别构转录因子(即，当小分子与别构转录因子结合时，别构转录因子与靶dsDNA的结合亲和力减弱，使得靶dsDNA从aTF-DNA复合体脱离)，当加入待测样品后，游离的dsDNA含量增加。因此，在一个优选实施方式中，为了减少背景噪音，提高本发明的灵敏度，先将别构转录因子与dsDNA混合形成aTF-dsDNA复合体，然后移除未结合的dsDNA，再加入待测样品。在这一实施方式中，由于加入待测样品之前，别构转录因子与dsDNA完全结合成aTF-dsDNA复合体，而基本上不存在游离的dsDNA，则加入待测样品后游离出来的dsDNA直接反映待测样品中的小分子的存在或含量。
在另一些实施方式中，所述别构转录因子为激活系统的别构转录因子(即，当小分子与别构转录因子结合时，别构转录因子与dsDNA的结合亲和力增强，使得dsDNA与别构转录因子结合成更多的aTF-dsDNA复合体)，当加入待测样品后，游离的dsDNA含量减少。本领域技术人员应理解的是，在这样的实施方式中，在步骤(1)之前应确定加入的dsDNA的量，由计算出减少的游离dsDNA间接反映待测样品中的小分子的存在或含量。在一些实施方式中，可在将dsDNA与别构转录因子混合之前、同时或之后加入待测样品。
在一个优选的实施方式中，所述别构转录因子是可固定在介质上的别构转录因子。因此，在步骤(1)中可进一步包括加入介质，例如，当所述可固定在介质上的别构转录因子是与纤维素结构域融合表达的别构转录因子时，所述介质为微晶纤维素。优选地，在加入待测样品和dsDNA之前，使别构转录因子与介质预先结合，将别构转录因子固定在介质上，便于后续操作。例如，将可商购的介质(例如微晶纤维素)与可固定在介质上的别构转录因子(例如与纤维素结构域融合表达的别构转录因子)混合并孵育，进行混合和孵育的具体条件可由本领域技术人员根据实际需要选择，只要保证与可固定在介质上的别构转录因子可充分固定在所述介质上即可，例如室温反应5-15min(但不限于此)。优选地，在加入待测样品和dsDNA之前，将未固定的别构转录因子除去。进一步，可对固定在介质上的别构转录因子的含量或浓度进行测定，例如使用本领域已知的蛋白质浓度检测方法(如使用Bradford分析试剂盒)。
步骤(2)中将游离dsDNA与步骤(1)中的蛋白质、小分子化合物和核酸混合物分离的方法是本领域熟知的，例如包括但不限于离心、沉降、磁珠法、层析法、亲和柱法等等。本领域技术人员可以根据实际情况选择具体的分离方法和参数，这一点并不会对本发明进行限制。在所述别构转录因子是可固定在介质上的别构转录因子的实施方式中，仅仅通过过滤或离心(例如室温下，7000rpm)即可将游离的dsDNA片段分离，操作简单、节约时间和成本，在经济上是可行的。也可省略步骤(2)的分离步骤，直接在步骤(1)的混合物中加入CRISPR/Cas12a系统和任选的ssDNA探针。
步骤(3)中，采用本领域中常用的检测荧光信号或吸收光信号的方法和仪器来检测产生的光信号。例如，当产生的信号为荧光信号时，采用酶标仪来测量。应当指出的是，当对所需检测的样品或小分子化合物进行定性分析时，产生的颜色变化(如上文所述，包括在“吸收光信号”的范围内)可不进行量化分析，而仅仅进行比色分析，这一点也在本发明请求保护的范围内。
步骤(4)中，可基于参比水平，对所产生的信号进行分析，从而对所述小分子的存在或含量进行检测。所述“参比水平”在本发明中与“参比样品”、“参照水平”可互换使用，是指条件的对照。例如，在 对小分子进行定性检测(检测存在或不存在)的上下文中，参比水平可以是不含所述小分子的样品的水平。在对小分子进行定量检测(检测含量)的上下文中，参比水平可以是含有已知量的小分子的样品的水平。就对包含小分子的样本的存在和含量进行确定而言，参比水平是参照值，能够将样品归一化至合适的标准，以推断样品中小分子的存在、不存在或含量。在一些实施方式中，参比水平可以是之前确定的水平，例如可以是预先确定的数量或比例，而不需要以本文所述的检测方法的相同的物理迭代进行确定。
在本发明中，所述样品以及所述试剂可处于溶液形式，所述溶液只要不影响aTF与效应物/dsDNA结合和CRISPR/Cas12a的切割即可。本领域技术人员可根据实际应用或需求对其进行选择或调整。
试剂盒
本发明还提供了一种检测小分子的试剂盒，所述试剂盒包含本发明所述的生物传感器以及检测元件。在本文中，所述检测元件是指对本发明的生物传感器产生的光信号进行检测的元件、设备或系统。在一些实施方式中，所述检测元件用于通过荧光分析或吸收光分析(包括比色分析)对所述DNA探针产生的光信号进行分析，从而获得对小分子的定性或定量检测结果。
另一方面，本发明还提供了一种检测小分子的试剂盒，所述试剂盒包含本发明所述的试剂以及检测试剂。在本发明中，所述检测试剂是指对使用本发明的试剂所产生的光信号进行检测的试剂。在一些实施方式中，所述检测试剂用于通过荧光分析或吸收光分析(包括比色分析)对所述DNA探针产生的光信号进行分析，从而获得对小分子的定性或定量检测结果。
在一些实施方式中，本发明所述的试剂盒进一步包含使用本发明的生物传感器和/或试剂检测小分子的递送工具或装置(例如移液枪)、洗涤缓冲液、稀释缓冲液、终止缓冲液(例如，用于终止显色)、微滴定板(例如98孔或384孔，用于进行反应和检测)、一个或多个容器、记载使用说明的数据载体(例如说明书或计算机可读介质)、标准品(例如含有已知量的小分子的样品)，以及它们的组合等等。
本文所述各方面的实施方式可由如下编号的段落说明：
1.一种用于检测小分子的组合物，所述组合物包含识别试剂和换能试剂，其中，
所述识别试剂包含别构转录因子(aTF)和双链DNA(dsDNA)；以及
所述换能试剂包含CRISPR/Cas12a系统，其中，所述CRISPR/Cas12a系统具有向导RNA，
其中，所述dsDNA包含所述aTF的结合位点、所述CRISPR/Cas12a系统的识别位点PAM以及与所述向导RNA至少部分互补的序列，
其中，所述组合物中还含有发光/生色基团，所述发光/生色基团连接至能够被所述CRISPR/Cas12a系统切割的核酸。
2.如段落1所述的组合物，其中，所述换能试剂进一步包含单链DNA(ssDNA)。
3.如段落1或2所述的组合物，其中，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)。
4.如段落1-3任一段所述的组合物，其中，所述dsDNA为激活dsDNA。
5.如段落1-4任一段所述的组合物，其中，所述dsDNA为dsDNA探针，并具有发光/生色基团及其淬灭基团。
6.如段落2所述的组合物，其中，所述ssDNA为ssDNA探针，并具有发光/生色基团及其淬灭基团。
7.如段落5或6所述的组合物，其中，所述dsDNA探针或所述ssDNA探针的两端缀合有发光/生色基团及其淬灭基团。
8.如段落1-7中任一段所述的组合物，其中，所述小分子具有50-5000道尔顿、优选50-1000道尔顿的分子量。
9.如段落1-8中任一段所述的组合物，其中，所述小分子为重金属离子、毒素、药物、代谢物、污染物或上述物质的分解产物。
10.如段落1-9中任一段所述的组合物，其中，所述小分子的存在通过所述aTF与所述dsDNA触发CRISPR/Cas12a的切割活性。
11.如段落1-10中任一段所述的组合物，其中，所述转录因子结合位点的长度为10-40bp、优选15-25bp、更优选17-19bp。
12.如段落1-11中任一段所述的组合物，其中，所述识别试剂包含可固定在介质上的别构转录因子和/或可固定在介质上的dsDNA。
13.如段落1-12中任一段所述的组合物，其中，所述识别试剂包含可固定在介质上的别构转录因子。
14.如段落1-13中任一段所述的组合物，其中，所述介质选自于由如下所组成的组：硝化纤维素膜或尼龙膜、亲和柱层析基质、磁珠、固体填料、微晶纤维素、或可商购的蛋白质固定化介质。
15.如段落13所述的组合物，其中，所述识别试剂包含可固定在微晶纤维素上的与微晶纤维素结构域融合表达的别构转录因子。
16.如段落1-15中任一段所述的组合物，其中，所述dsDNA的长度为20-80bp、更优选55-65bp，最优选58-60bp。
17.如段落1-16中任一段所述的组合物，其中，所述识别试剂和所述换能试剂以单独的形式提供并且在不同的空间进行反应。
18.如段落6-17中任一段所述的组合物，其中，所述ssDNA探针是长度为10-30bp任意单链DNA
19.如段落6-17中任一段所述的组合物，其中，所述ssDNA探针是长度为20bp的任意单链DNA。
20.如段落1-12中任一段所述的组合物，其中，所述发光/生色基团选自于由如下所组成的组：发光有机化合物、发光无机化合物、荧光团、纳米颗粒、量子点和生色团，以及它们的组合。
21.如段落1所述的组合物，其中，所述组合物包含识别试剂和换能试剂，其中：
所述识别试剂包含别构转录因子(aTF)和激活双链DNA(激活dsDNA)，所述激活dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列，以及
所述换能试剂包含所述CRISPR/Cas12a系统和单链DNA探针(ssDNA探针)，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)，所述ssDNA探针两端分别缀合有发光/生色基团及其淬灭基团；
22.如段落1所述的组合物，其中，所述组合物包含识别试剂和换能试剂，其中：
所述识别试剂包含aTF和双链DNA探针(dsDNA探针)，所述dsDNA探针包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA(gRNA)至少部分互补的序列，并且所述dsDNA探针两端分别缀合有发光/生色基团及其淬灭基团，以及
所述换能试剂包含CRISPR/Cas12a系统，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述gRNA。
23.如段落1所述的组合物，其中，所述组合物处于生物传感器的形式。
24.如段落23所述的组合物，其中，所述生物传感器包含识别元件和换能元件，其中，
所述识别元件包含别构转录因子(aTF)和双链DNA(dsDNA)，所述dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列；以及所述换能元件包含所述CRISPR/Cas12a系统，
其中，所述生物传感器中还含有发光/生色基团。
25.如段落24所述的组合物，其中，所述换能元件进一步包含单链DNA(ssDNA)。
26.如段落24或25所述的组合物，其中，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)。
27.如段落24-26任一段所述的组合物，其中，所述dsDNA为激活dsDNA。
28.如段落24-26任一段所述的组合物，其中，所述dsDNA为dsDNA探针，并具有发光/生色基团及其淬灭基团。
29.如段落25所述的组合物，其中，所述ssDNA为ssDNA探针，并具有发光/生色基团及其淬灭基团。
30.如段落28或29所述的组合物，其中，所述dsDNA探针或所述ssDNA探针的两端缀合有发光/生色基团及其淬灭基团。
31.如段落24所述的组合物，其中，所述识别元件包含别构转录因子(aTF)和激活双链DNA(激活dsDNA)，所述激活dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点(即，原型间隔区邻近基序，PAM)以及与向导RNA至少部分互补的序列，以及
所述换能元件包含所述CRISPR/Cas12a系统以及单链DNA探针(ssDNA探针)，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)，所述ssDNA探针两端分别缀合有发光/生色基团及其淬灭基团；
32.如段落24所述的组合物，其中，所述识别元件包含别构转录因子(aTF)和双链DNA探针(dsDNA探针)，所述dsDNA探针包含所述别构转录因子的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA(gRNA)至少部分互补互补的序列，并且所述dsDNA探针两端分别缀合有发光/生色基团及其淬灭基团，以及
所述换能元件包含所述CRISPR/Cas12a系统，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述gRNA。
33.如段落1-32中任一段所述的组合物，其中，所述识别试剂和所述换能试剂以单独的形式提供并且在不同的空间进行反应。
34.如段落21所述的组合物，其中，所述别构转录因子和dsDNA，所述CRISPR/Cas12a蛋白、向导RNA(gRNA)和ssDNA探针作为单一试剂提供；或者
所述别构转录因子和激活dsDNA，所述CRISPR/Cas12a蛋白、向导RNA(gRNA)和ssDNA探针作为单独的试剂提供。
35.如段落22所述的组合物，其中，所述别构转录因子和dsDNA探针，所述CRISPR/Cas12a蛋白和向导RNA(gRNA)作为单一试剂提供；或者
所述别构转录因子和dsDNA探针，所述CRISPR/Cas12a蛋白和向导RNA(gRNA)作为单独的试剂提供。
36.一种用于检测小分子的试剂盒，所述试剂盒包含如段落1-35中任一段所述的组合物。
37.如段落36所述的试剂盒，其中，所述试剂盒还包含用于对光信号进行检测的检测装置。
38.如段落38或39所述的试剂盒，其中，所述试剂盒还包含操作如段落1-35中任一段所述的生物传感器时所用的递送工具或装置、洗涤缓冲液、稀释缓冲液、终止缓冲液、记载使用说明的数据载体、标准品或容器，以及它们的组合。
39.一种对待测样品中的小分子进行检测的方法，所述方法包括用段落1-35中任一段所述的组合物或如段落36-38中任一段所述的试剂盒对所述待测样品中的小分子进行检测。
40.如段落39所述的方法，其中，所述方法包括：
(1)将所述待测样品与识别试剂进行混合并孵育；(2)从所得的混合物中分离游离的dsDNA片段；(3)将分离的游离dsDNA加入换能试剂中，并检测所产生的光信号；以及，(4)基于所产生的信号来检测所述小分子的存在或含量。
41.如段落39或40所述的方法，其中，所述方法包括：
(1)将所述待测样品、aTF和dsDNA进行混合并孵育；(2)从所得的混合物中分离游离的dsDNA片段；(3)将分离的游离dsDNA加入CRISPR/Cas12a蛋白和向导RNA中，并检测所产生的光信号；以及(4)基于所产生的信号来检测所述小分子的存在或含量。
42.如段落39或40所述的方法，其中，所述方法包括：
(1)将所述待测样品、aTF和激活dsDNA片段进行混合并孵育；
(2)从步骤(1)中的混合物分离游离的激活dsDNA片段；
(3)向步骤(2)中分离的游离激活DNA片段中加入CRISPR/Cas12a蛋白、向导RNA和ssDNA探针，并检测所产生的光信号；以及
(4)基于所产生的信号，对所述小分子的存在或含量进行检测。
43.如段落39或40所述的方法，其中，所述方法包括：
(1)将所述待测样品、aTF和dsDNA探针进行混合并孵育；
(2)从步骤(1)中的混合物分离游离的dsDNA探针；
(3)向步骤(2)中分离的游离dsDNA探针中加入CRISPR/Cas12a蛋白和向导RNA，并检测所产生的光信号；以及
(4)基于所产生的信号，对所述小分子的存在或含量进行检测。
44.如段落42或43所述的方法，其中，在步骤(1)中，所述别构转录因子和所述激活dsDNA的摩尔比为≥5:1；所述别构转录因子和所述dsDNA探针的摩尔比为≥5:1。
45.如段落42或43所述的方法，其中，在步骤(1)中，先将所述别构转录因子与所述激活dsDNA混合形成复合体，然后移除未结合的激活DNA，再加入所述待测样品；或者，先将所述别构转录因子与所述dsDNA探针混合形成复合体，然后移除未结合的是所述dsDNA探针，再加入所述待测样品。
46.如段落40-45中任一段所述的方法，其中，在步骤(1)中，所述混合是在室温下1min以上。
47.如段落40-46中任一段所述的方法，其中，在步骤(1)中，所述别构转录因子是可固定在介质上的别构转录因子。
48.如段落47所述的方法，其中，在步骤(1)之前，加入介质使所述可固定在介质上的别构转录因子固定至介质上。
49.如段落39-48中任一段所述的方法，其中，所述待测样品来自环境、受试者、食品、饮用水、化妆品或饲料。
50.如段落49所述的方法，其中，所述待测样品选自以下中的一种或多种：地下水、中水、海水、废水、采矿废料；血液、血清、血浆、痰、脑脊液流体、尿液、泪液、肺泡分离物、胸膜液、囊液、组织、唾液。
51.如段落40-50所述的方法，其中，在步骤(1)之前，对所述待测样品进行富集、提取和/或纯化的预处理。
52.如段落40-51中任一段所述的方法，其中，在步骤(2)中，通过过滤、离心、沉降、磁珠法、层析法、亲和柱法进行分离。
53.如段落40-52中任一段所述的方法，其中，在步骤(3)中，通过荧光分析或比色分析进行检测。
54.如段落40-53中任一段所述的方法，其中，在步骤(4)中，基于参比水平，对所述光信号进行分析，所述参比水平为不含所述小分子的样品的水平或含有已知量的小分子的样品的水平。
55.段落1-35中任一段所述的组合物在制备检测小分子的试剂盒中的用途。
56.如段落55所述的用途，其中，所述试剂盒用于环境污染监控、食品和化妆品质量控制和疾病诊断。
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实施例1
以下实施例中使用的dsDNA片段(如SEQ ID NOs:5‐7所示的核苷酸序列)、向导RNA(如SEQ ID NOs:9‐11所示的核苷酸序列)、引物(表2所示)以及探针(如SEQ ID NO:8所示的核苷酸序列，5’端标记有FAM，3’端标记有BHQ)均由金斯瑞公司人工合成。
以下实施例中所使用的培养基和试剂如下：
LB培养基：1％NaCl、1％蛋白胨、0.5％酵母粉
LB平板：1％NaCl、1％蛋白胨、0.5％酵母粉、1.5％琼脂粉
结合缓冲液：50mM Tris-HCl(7.4)、500mM NaCl、20mM咪唑，2mM DTT、5％甘油
洗杂缓冲液：50mM Tris-HCl(7.4)、500mM NaCl、40mM咪唑，2mM DTT、5％甘油
洗脱缓冲液：50mM Tris-HCl(7.4)、500mM NaCl、500mM咪唑，2mM DTT、5％甘油
透析缓冲液：50mM Tris-HCl(7.4)、500mM NaCl、2mM DTT、5％甘油
离子交换平衡缓冲液：50mM Tris-HCl(7.4)、2mM DTT、5％甘油
离子交换洗脱缓冲液：50mM Tris-HCl(7.4)、1M NaCl、2mM DTT、5％甘油
Tris-HCl缓冲液：50mM Tris-HCl(7.4)、200mM NaCl
表2实施例1-实施例2中使用的引物
Figure PCTCN2020084111-appb-000003
Figure PCTCN2020084111-appb-000004
实施例1尿酸生物传感器
在本实施例中，利用别构转录因子HucR和激活dsDNA片段(dsDNA hucR；其中，包含HucR结合位点(与gRNA互补的序列包含在其中)和CRISPR/Cas12a识别位点(PAM))作为识别元件，以CRISPR/Cas12a、gRNA和单链DNA(ssDNA；两端标记有FAM荧光团和BHQ荧光淬灭团，作为探针)作为换能元件，构建能够感应尿酸的生物传感器平台。
HucR蛋白为来源于耐辐射奇球菌(Deinococcus radiodurans)的阻遏蛋白，其氨基酸序列可如SEQ ID NO:1所示。如图1所示，在无尿酸(UA)时，HucR能结合dsDNA hucR上的HucR作用位点形成HucR-dsDNA hucR复合体。当存在尿酸时，尿酸特异性地结合HucR，使dsDNA hucR从HucR释放获得游离形式的dsDNA hucR，游离形式的dsDNA hucR激活CRISPR/Cas12a系统，引起DNA探针切割，产生荧光信号。因此，可通过监控荧光信号确定样品中的尿酸浓度。
1.HucR-CBD融合蛋白的克隆、表达和纯化
以含有CBD基因(编码的氨基酸序列如SEQ ID NO:4所示)的pET35b质粒(武汉淼灵生物科技有限公司)为模版，用引物CBD-F/HucR-CBD-R扩增获得CBD片段；使用Xho I单酶切(反应体系：10x cutsmart buffer 2μL；质粒1μg；Xho I 1μL；H 2O补充至20μL；37℃反应1h)对pET23b-HucR质粒(预先构建：使用Nde I和xho I对pET23b质粒和HucR基因片段进行双酶切(反应体系：10x cutsmart buffer 2μL；质粒/HucR基因片段1μg；Xho I 1μL；Nde I 1μL；H 2O补充至20μL；37℃反应1h)，纯化回收后通过T4 DNA连接酶连接(反应体系：10x T4 ligase buffer 1μL；质粒50-100ng；HucR基因片段50-100ng；H 2O补充至10μL；22℃反应1h)所得)进行线性化处理；使用无缝克隆(单片段连接)试剂盒(Tolo Biotechnology，上海)，根据制造商提供的说明书用Exmax将CBD片段和线性化的pET23b-hucR进行组装，获得pET23b-HucR-CBD质粒，进行PCR和测序，确认为阳性质粒。采用钙转法将10ng质粒pET23b-HucR-CBD转入大肠杆菌BL21(天根生化科技有限公司，北京)，Amp抗性筛选阳性克隆。挑取单克隆菌落接种于100mL LB(Amp +)培养基中，37℃，200rpm摇床培养过夜(～12h)。取2.5mL(1v/v％接种量)培养液转接至250mL LB(Amp +)培养基，加入终浓度为0.2mM的IPTG，16℃、180rpm低温诱导过夜，随后在4℃、9,000rpm离心15min，收集菌体并用洗杂缓冲液洗涤菌体。每2L菌体用50mL预冷的结合缓冲液悬浮。将悬浮液置于冰上，超声裂解菌体(超声波细胞粉碎机，超声3s，间歇5s，30W，20min)，4℃、 12000rpm离心30min，取上清重复上述离心操作至澄清。将裂解物流经HisTrap FF柱(GE Healthcare)上，使用洗脱缓冲液进行洗脱。收集峰值级分并且进行透析(透析膜来自上海生工；透析缓冲液)。然后将透析后的溶液上样至HiTrap QHP柱(GE Healthcare)，用洗脱缓冲液进行洗脱。收集峰值级分并使用超滤管(milipore)进行浓缩。将浓缩液上样至HiLoad 16/600 Superdex 200pg柱进行快速蛋白液相色谱(FPLC；AKTA Exporer 100，GE Healthcare)。通过SDS-PAGE对纯化的HucR-CBD蛋白进行验证并且使用Bradford方法测定其浓度为3mg/mL。
2.HucR-CBD融合蛋白的活性分析
因CBD结构域与HucR分子量相近，为了分析CBD结构域对HucR蛋白的活性影响，利用凝胶阻滞实验(EMSA)对HucR-CBD与dsDNA HucR的结合进行分析。使用引物dsDNA HucR-1和dsDNA HucR-2退火(95℃预变性5min；95℃变性30s，75个循环，每个循环降低1℃)得到60bp大小的dsDNA (HucR)，用于EMSA分析。EMSA的实验条件和数据采集方法均根据Wang等，Molecular Microbiology，2011，82(1)：236-250。将0、25、50、100、200nM的HucR-CBD与40nM dsDNA HucR混匀(处于20mL的缓冲液中：10mM Tris-HCl(pH7.5)，100mM KCl，1mM EDTA，0.1mM DTT，5％v/v甘油，0.01mg/ml牛血清白蛋白，dsDNA与aTF的结合以及效应物与dsDNA和aTF的作用均在此种缓冲液中进行)，在室温下避光反应15min，1.5％琼脂糖胶进行电泳，检测融合蛋白HucR-CBD与dsDNA HucR的结合。
结果如图2B所示，当HucR-CBD:dsDNA HucR＝200nM:40nM(即5:1)时，dsDNA HucR可被HucR-CBD完全结合，可见与CBD结构域融合表达不影响HucR的功能。
3.HucR-CBD融合蛋白对尿酸的识别与响应
为了分析效应物尿酸对HucR-CBD-dsDNA HucR复合物的解离作用，向HucR-CBD:dsDNA HucR＝5:1的孵育体系中加入不同浓度的尿酸(0、0.05、0.5、5、50、500μM)，通过EMSA检测dsDNA HucR的解离。结果如图2C所示，随着尿酸浓度的增加，游离的dsDNA HucR不断增加。
4.Cas12a蛋白和gRNA的制备
构建pET28TEV-Cas12a质粒(LbCas12a，吐露港公司)，以与表达和纯化HucR-CBD融合蛋白步骤的相同的步骤，将pET28TEV-Cas12a质粒转化至大肠杆菌BL21感受态细胞，0.2mM IPTG诱导蛋白表达后，离心收集菌体并经超声波破碎仪超声破碎后收集细胞裂解物，进行纯化和浓缩，得到浓度为1mg/mL的Cas12a纯化蛋白。
在Taq DNA聚合酶缓冲液中使T7引物与crRNA-HucR引物退火(20μL体系：10x Taq DNA聚合酶缓冲液2μL；T7引物9μL；crRNA-HucR引物9μL；PCR程序：95℃预变性5min；95℃变性30s，每个循环降低1℃，70个循环)合成crRNA(即gRNA)的模板。然后用HiScribeTM T7快速高效RNA合成试剂盒(NEB)转录成crRNA并用RNA Clean&Concentrator TM-5(Zymo Research)进行纯化。使用NanoDrop2000分光光度仪(Thermo Fisher Scientific)测量所得的crRNA的浓度，为2000-3000ng/μL。
5.对尿酸的检测
取2mg微晶纤维素(上海生工；货号9004-34-6)用Tris-HCl缓冲液洗涤两次，与融合蛋白HucR-CBD(200nM)混合于NEB
Figure PCTCN2020084111-appb-000005
缓冲液，室温混合孵育10分钟。室温、7000rpm离心后弃上清，使用NEB
Figure PCTCN2020084111-appb-000006
缓冲液洗涤3次除去未结合的蛋白，使用Bradford分析试剂盒(天根生化科技公司)对固定化的蛋白质进行定量；加入100nM dsDNA HucR，室温反应～9分钟，室温、7000rpm离心弃上清，洗涤≥1次除去未结合的dsDNA HucR；在所得到的dsDNA-蛋白-纤维素复合物中各加入500μM尿酸及其类似物(腺嘌呤、鸟嘌呤、次黄嘌呤)，室温反应15min，室温、7000rpm离心取上清。将上清液(其中可能包含游离形式的dsDNA HucR，以40nM游离形式的dsDNA HucR作为阳性对照)加入Cas12a、gRNA以及经FAM/BHQ修饰的ssDNA(序列如SEQ ID NO:9所示)的混合物(50nM Cas12a、50nM gRNA以及250nM ssDNA，处于20μL NEB
Figure PCTCN2020084111-appb-000007
缓冲液中)，混合后立即置于BMG CLARIOstar酶标仪(BMG Labtech，英国)中37℃反应，在480nm激发光和520nm发射光下测定并记录反应体系的荧光强度。结果如图3C所示，阳性对照(即游离形式的dsDNA HucR)和尿酸组获得了最强的荧光强度，而尿酸类似物(腺嘌呤、鸟嘌呤、次黄嘌呤)组的荧光强度小于阳性对照组的5％。并且该结果与EMSA的结果(图3B)类似，说明本发明的尿酸生物传感器能够准确地对小分子进行识别并且进行检测。
6.对生物传感器性能的分析
6.1 dsDNA线性检测范围
为了探究不同浓度的dsDNA对荧光强度变化的影响，取不同浓度的dsDNA HucR(1、5、10、25、50、100pM)加入Cas12a、gRNA以及经FAM/HQ修饰的ssDNA(序列如SEQ ID NO:9所示)的混合物(50nM Cas12a、50nM gRNA以及250nM ssDNA，处于20μL NEB
Figure PCTCN2020084111-appb-000008
缓冲液中)，混合后立即置于BMG CLARIOstar酶标仪(BMG Labtech，英国)中37℃反应，在480nm激发光和520nm发射光下测定并记录反应体系的荧光强度。结果如图4A(左栏)所示，随着dsDNA的浓度不断增加，反应体系的荧光强度也随之增加。为分析dsDNA与反应体系荧光强度变化之间的线性关系，取荧光值线性增加区间的斜率，以dsDNA浓度为横坐标，对应的斜率为纵坐标作图4A(右栏)，由此可知dsDNA的线性检测范围1-25pM。
6.2尿酸的线性检测范围
为了探究不同浓度的尿酸对荧光强度变化的影响，取不同浓度的尿酸以如上的方法进行检测，测定并记录反应体系荧光强度，结果如图4B(左栏)所示，随着尿酸浓度的增加，游离的dsDNA含量随之增多，荧光强度也随之提高。反应体系的荧光强度的变化随着尿酸的增加而逐渐增加，为分析尿酸浓度与反应体系荧光强度变化间的线性关系，取荧光值线性增加区间的斜率，以尿酸浓度为横坐标，对应的斜率为纵坐标作图4B(右栏)，可知尿酸的线性检测范围25-500nM，回归方程为：y＝10.7x+1289.8，R 2＝0.992。本实施例传感器对尿酸的检测极限为nM级，而现有的尿酸生物传感器通常为μM级(如专利201810224843.8中所记载的尿酸生物传感器，1.71μM)，可见本发明的生物传感器具有出色的检测灵敏度(可低至25nM)。
6.3对临床血液样品的分析
取等量的血清和氯仿混合并且剧烈震荡，室温、10,000g离心10分钟，收集上清液。使用如上的方法采用本实施例的尿酸生物传感器对血清样品中的尿酸进行检测。为了避免吸取的时候的误差，用水将血清样品上清液稀释10倍，使用1μL稀释液进行检测。由于HPLC能够非常精确的检测血清中的尿酸含量，将样品进行HPLC作为对照。HPLC条件：Agilent SB-Aq柱(4.6mm×150mm，5μm，1mL/min)；水作为洗脱液，在284nm处检测(UV检测器；Agilent 1260 infinity II LC系统)。将由HPLC测得的尿酸浓度为纵坐标，本实施例的生物传感器检测方法得到的尿酸浓度为纵坐标做图(图5A)，可见两种方法测得的结果线性相关(y＝0.978x，R 2＝0.993)，这说明本发明的检测方法具有较高的可信度。
进一步，将本实施例的检测方法与临床上使用的Backman尿酸检测试剂盒进行对比，同样将Backman测得的尿酸浓度为纵坐标，本实施例的生物传感器检测方法得到的尿酸浓度为纵坐标做图(图5B)，可见两种方法测得的结果线性相关(y＝1.023x，R 2＝0.983)，也说明了本发明的检测方法具有较高的可信度。
人体血清中尿酸的正常范围为166.4μM-546.7μM，而由于本发明的生物传感器非常灵敏，仅需要非常少量的血液样品(约1μL)即可得到准确的结果(图5C)，使得反应体系体积较小，可以在96孔板甚至384孔板上进行该检测。并且，本发明的方法仅需15-25分钟，至多25分钟即可得到结果，不仅节约时间，而且成本低、操作简便，非常适合用于对血液等样品中的尿酸进行高通量检测。
6.4与其它检测方法的比较
为了进一步评估本实施例的尿酸生物传感器的优点，将其与现有技术中已报道的各种检测方法进行对比，并且将结果汇总与表3中。
表3.不同小分子检测方法的比价
Figure PCTCN2020084111-appb-000009
Figure PCTCN2020084111-appb-000010
RT-qPCR：实时荧光定量PCR；RCA：滚环扩增；RPA：重组酶聚合酶扩增；PGM：血糖仪；ISDA：等温链置换扩增；临床方法：Backman试剂盒；CaT-SMelor：本发明的生物传感器；Y：可适用；N：不可适用；NA：未获得。
而且，对于本实施的尿酸生物传感器，由于荧光信号相对稳定，因此该生物传感器的荧光半衰期超过200min，这为研究员或其他使用者提供更多的时间记录和分析实验结果。
综上所述，本发明的尿酸生物传感器具有出色的检测灵敏度、较高的可信度、节约时间和成本，操作简单，非常适合于需要高通量、高速、高灵敏度的小分子体外检测，在实验室以及医疗、工业应用中均具有广阔的应用前景。
实施例2四环素生物传感器和对羟基苯甲酸(p-HBA)传感器
为了验证本发明传感器和方法的普适性，采用实施例1的理念，设计了分别响应于四环素和p-HBA的传感器。响应于四环素的生物传感器利用别构转录因子TetR和双链激活DNA片段(dsDNA TetR；其中包含TetR结合位点和CRISPR/Cas12a识别位点(PAM))作为识别元件，以CRISPR/Cas12a、gRNA和单链DNA(ssDNA；两端标记有FAM荧光团和BHQ荧光淬灭团，作为探针)作为换能元件。响应于p-HBA的生物传感器利用别构转录因子HosA和双链激活DNA片段(dsDNA HosA；其中包含HosA结合位点和CRISPR/Cas12a识别位点(PAM))作为识别元件，以CRISPR/Cas12a、gRNA和单链DNA(ssDNA；两端标记有FAM荧光团和BHQ荧光淬灭团，作为探针)作为换能元件。
根据实施例1的方法和步骤，制备TetR-CBD融合蛋白和HosA-CBD融合蛋白，构建四环素生物传感器和p-HBA传感器，并对p-HBA传感器的性能进行分析。
1.对p-HBA传感器性能的分析
1.1 HosA-CBD融合蛋白活性分析
将不同浓度(0、25、50、100、200nM)的HosA-CBD与40nM的dsDNA HosA(核苷酸序列如SEQ ID NO:6所示)混合，室温反应15min，进行EMSA检测HosA-CBD与dsDNA HosA的结合。结果如图2D所示，当HosA-CBD:dsDNA HosA＝5:1时，dsDNAHosA可被HosA-CBD完全结合，与CBD结构域融合表达不影响HosA的功能活性。
1.2融合蛋白HosA-CBD对p-HBA的识别与响应
为了分析效应物p-HBA对HosA-CBD和dsDNA HosA复合物的解离作用，向HosA-CBD:dsDNA HosA＝5:1的反应体系中加入不同浓度(0、0.18、1.8、18、180、1800μM)的p-HBA通过EMSA检测dsDNA HosA的解离。结果如图2E所示，随着p-HBA浓度的增加，游离的dsDNA HosA不断增加。
1.3对p-HBA的检测
取2mg微晶纤维素(上海生工；货号9004-34-6)用Tris-HCl缓冲液洗涤两次，与融合蛋白HosA-CBD(200nM)混合于NEB
Figure PCTCN2020084111-appb-000011
缓冲液，室温混合孵育10分钟。室温、7000rpm离心后弃上清，使用NEB
Figure PCTCN2020084111-appb-000012
缓冲液洗涤3次除去未结合的蛋白，使用Bradford分析试剂盒(天根生化科技公司)对固定化的蛋白质进行定量；加入100nM dsDNA HosA，室温反应10分钟，室温、7000rpm离心弃上清，洗涤3次除去未结合的dsDNA HosA；在所得到的dsDNA-蛋白-纤维素复合物中各加入1.8mM p-HBA及其类似物(对羟苯基乙醇、对氨基苯甲酸(p-ABA)、对羟基苯甲酸甲酯(p-MHB)、对羟基苯甲醇(p-HBnOH)，室温反应15min，室温、7000rpm离心取上清。将上清液(其中可能包含游离形式的dsDNA HosA，以1.8mM游离形式的dsDNA HosA作为阳性对照)加入Cas12a、gRNA(核苷酸序列如SEQ ID NO:10所示)以及经FAM/BHQ的ssDNA(序列如SEQ ID NO:9所示)的混合物(50nM Cas12a、50nM gRNA以及250nM ssDNA，处于20μL NEB
Figure PCTCN2020084111-appb-000013
缓冲液中)，混合后立即置于BMG CLARIOstar酶标仪(BMG Labtech，英国)中37℃反应，在480nm激发光和520nm发射光下测定并记录反应体系的荧光强度。结果如图3E所示，阳性对照(即游离形式的dsDNA HosA)和p-HBA组获得了最强的荧光强度，而p-HBA类似物(对羟苯基乙醇、p-ABA、p-MHB、p-HBnOH)组的荧光强度小于阳性对照组的5％。并且该结果与EMSA的结果(图3D)类似，说明本发明的p-HBA生物传感器能够准确地对小分子进行识别并且进行检测。
1.4 p-HBA的线性检测范围
为了探究不同浓度的p-HBA对荧光强度变化的影响，取不同浓度(1.8、9、18、90、180、900、1800nM)的p-HBA进行检测，测定并记录反应体系荧光强度，结果如图4C(左栏)所示，随着p-HBA浓度的增加，游离的dsDNA HosA含量随之增多，荧光强度的变化也随之提高。为分析p-HBA浓度与反应体系荧光强度变化之间的线性关系，取荧光值线性增加区间的斜率，以p-HBA浓度为横坐标，对应的斜率为纵坐标作图4C(右栏)，可知dsDNA的线性检测范围9-180nM，回归方程分别为：y＝6.3x+656.6，R 2＝0.992。因此，根据本发明的PHBA传感器性能也具有出色的检测极限(可低至1.8nM)。
因此，本发明的传感器和方法具有普适性，可借助多种别构转录因子对特定小分子化合物进行高灵敏度、高速、高通量的检测，不仅操作简单，而且成本低廉，在实验室以及工业应用中均具有广阔的应用前景。

Claims (47)

  1. 一种用于检测小分子的组合物，所述组合物包含识别试剂和换能试剂，其中，
    所述识别试剂包含别构转录因子(aTF)和双链DNA(dsDNA)；以及
    所述换能试剂包含CRISPR/Cas12a系统，其中，所述CRISPR/Cas12a系统具有向导RNA，
    其中，所述dsDNA包含所述aTF的结合位点、所述CRISPR/Cas12a系统的识别位点PAM以及与所述向导RNA至少部分互补的序列，
    其中，所述组合物还含有发光/生色基团，所述发光/生色基团连接至能够被所述CRISPR/Cas12a系统切割的核酸。
  2. 如权利要求1所述的组合物，其中，所述能够被所述CRISPR/Cas12a系统切割的核酸为所述dsDNA，所述dsDNA作为dsDNA探针，并具有发光/生色基团及其淬灭基团。
  3. 如权利要求1所述的组合物，其中，所述能够被所述CRISPR/Cas12a系统切割的核酸为单链DNA(ssDNA)，所述ssDNA作为ssDNA探针，并具有发光/生色基团及其淬灭基团；其中，所述dsDNA为激活dsDNA。
  4. 如权利要求1-3中任一项所述的组合物，其中，所述CRISPR/Cas12a系统包含Cas12a蛋白与所述向导RNA(gRNA)。
  5. 如权利要求2或3所述的组合物，其中，所述dsDNA探针或所述ssDNA探针的两端缀合有发光/生色基团及其淬灭基团。
  6. 如权利要求1-5中任一项所述的组合物，其中，所述小分子具有50-5000道尔顿、优选50-1000道尔顿的分子量。
  7. 如权利要求1-6中任一项所述的组合物，其中，所述dsDNA中与所述向导RNA至少部分互补的序列与所述RNA具有至少70％以上的互补性。
  8. 如权利要求1-7中任一项所述的组合物，其中，所述别构转录因子选自来源于以下的别构转录因子中的一种或多种：耐辐射奇球菌、大肠杆菌、铜绿假单胞菌、霍乱弧菌、乳酸乳球菌、假单胞菌转座子、金黄色葡萄球菌、海洋沉积物芽孢杆菌、龟裂链霉菌、丁香假单胞菌、天蓝色链霉菌、不动杆菌属、谷氨酸棒状杆菌、嗜麦芽寡养单胞菌、和阿维链霉菌。
  9. 如权利要求1-8中任一项所述的组合物，其中，所述小分子为重金属离子、毒素、药物、代谢物、污染物或上述物质的分解产物。
  10. 如权利要求1-9中任一项所述的组合物，其中，所述小分子的存在通过所述aTF与所述dsDNA来触发CRISPR/Cas12a系统的切割活性。
  11. 如权利要求1-10中任一项所述的组合物，其中，所述转录因子结合位点的长度为10-40bp、优选15-25bp、更优选17-19bp。
  12. 如权利要求1-11中任一项所述的组合物，其中，所述别构转录因子和/或dsDNA固定在介质上。
  13. 如权利要求1-12中任一项所述的组合物，其中，所述别构转录因子固定在介质上。
  14. 如权利要求1-13中任一项所述的组合物，其中，所述介质选自于由如下所组成的组：硝化纤维素膜或尼龙膜、亲和柱层析基质、磁珠、固体填料、微晶纤维素、或可商购的蛋白质固定化介质。
  15. 如权利要求13所述的组合物，其中，所述别构转录因子与纤维素结构域(CBD)融合表达，并借由所述纤维素结构域固定在微晶纤维素上。
  16. 如权利要求1-15中任一项所述的组合物，其中，所述dsDNA的长度为20-80bp、更优选55-65bp，最优选58-60bp。
  17. 如权利要求1-16中任一项所述的组合物，其中，所述识别试剂和所述换能试剂以单独的形式提供并且在不同的空间进行反应。
  18. 如权利要求4-17中任一项所述的组合物，其中，所述ssDNA探针是长度为10-30bp任意单链DNA。
  19. 如权利要求4-17中任一项所述的组合物，其中，所述ssDNA探针是长度为20bp的任意单链DNA。
  20. 如权利要求1-12中任一项所述的组合物，其中，所述发光/生色基团选自于由如下所组成的组：发 光有机化合物、发光无机化合物、荧光团、纳米颗粒、量子点和生色团，以及它们的组合。
  21. 如权利要求3所述的组合物，其中，所述组合物包含识别试剂和换能试剂，其中：
    所述识别试剂包含别构转录因子(aTF)和激活双链DNA(激活dsDNA)，所述激活dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列，以及
    所述换能试剂包含所述CRISPR/Cas12a系统和单链DNA探针(ssDNA探针)，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述向导RNA(gRNA)，所述ssDNA探针两端分别缀合有发光/生色基团及其淬灭基团。
  22. 如权利要求2所述的组合物，其中，所述组合物包含识别试剂和换能试剂，其中：
    所述识别试剂包含aTF和双链DNA探针(dsDNA探针)，所述dsDNA探针包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA(gRNA)至少部分互补的序列，并且所述dsDNA探针两端分别缀合有发光/生色基团及其淬灭基团，以及
    所述换能试剂包含CRISPR/Cas12a系统，所述CRISPR/Cas12a系统包含CRISPR/Cas12a蛋白与所述gRNA。
  23. 如权利要求1-22中任一项所述的组合物，其中，所述组合物处于生物传感器的形式。
  24. 如权利要求23所述的组合物，其中，所述生物传感器包含识别元件和换能元件，其中，
    所述识别元件包含别构转录因子(aTF)和双链DNA(dsDNA)，所述dsDNA包含所述aTF的结合位点、CRISPR/Cas12a系统的识别位点PAM以及与向导RNA至少部分互补的序列；以及所述换能元件包含所述CRISPR/Cas12a系统，
    其中，所述生物传感器中还含有发光/生色基团，所述发光/生色基团连接至能够被所述CRISPR/Cas12a系统切割的核酸。
  25. 如权利要求21所述的组合物，其中，所述别构转录因子和dsDNA，所述CRISPR/Cas12a蛋白、向导RNA(gRNA)和ssDNA探针作为单一试剂提供；或者
    所述别构转录因子和激活dsDNA，所述CRISPR/Cas12a蛋白、向导RNA(gRNA)和ssDNA探针分别作为单独的试剂提供。
  26. 如权利要求22所述的组合物，其中，所述别构转录因子和dsDNA探针，所述CRISPR/Cas12a蛋白和向导RNA(gRNA)作为单一试剂提供；或者
    所述别构转录因子和dsDNA探针，所述CRISPR/Cas12a蛋白和向导RNA(gRNA)分别作为单独的试剂提供。
  27. 一种用于检测小分子的试剂盒，所述试剂盒包含如权利要求1-26中任一项所述的组合物。
  28. 如权利要求27所述的试剂盒，其中，所述试剂盒还包含用于对光信号进行检测的检测装置。
  29. 如权利要求27或28所述的试剂盒，其中，所述试剂盒还包含操作如权利要求1-26中任一项所述的生物传感器时所用的递送工具或装置、洗涤缓冲液、稀释缓冲液、终止缓冲液、记载使用说明的数据载体、标准品或容器，以及它们的组合。
  30. 一种对待测样品中的小分子进行检测的方法，所述方法包括用权利要求1-26中任一项所述的组合物或如权利要求27-29中任一项所述的试剂盒对所述待测样品中的小分子进行检测。
  31. 如权利要求30所述的方法，其中，所述方法包括：
    (1)将所述待测样品与识别试剂进行混合并孵育；(2)从步骤(1)所得的混合物中分离游离的dsDNA片段；(3)将分离的游离dsDNA加入换能试剂中，并检测所产生的光信号；以及，(4)基于所产生的信号来检测所述小分子的存在或含量。
  32. 如权利要求30或31所述的方法，其中，所述方法包括：
    (1)将所述待测样品、aTF和dsDNA进行混合并孵育；(2)从所得的混合物中分离游离的dsDNA片段；(3)向步骤(2)中分离得到的游离dsDNA加入CRISPR/Cas12a蛋白和向导RNA中，并检测所产生的光信号；以及(4)基于所产生的信号来检测所述小分子的存在或含量。
  33. 如权利要求30或31所述的方法，其中，所述方法包括：
    (1)将所述待测样品、aTF和激活dsDNA片段进行混合并孵育；
    (2)从步骤(1)所得的混合物中分离游离的激活dsDNA片段；
    (3)向步骤(2)中分离得到的游离的激活dsDNA片段中加入CRISPR/Cas12a蛋白、向导RNA和ssDNA探针，并检测所产生的光信号；以及
    (4)基于所产生的信号，对所述小分子的存在或含量进行检测。
  34. 如权利要求30或31所述的方法，其中，所述方法包括：
    (1)将所述待测样品、aTF和dsDNA探针进行混合并孵育；
    (2)从步骤(1)中的混合物分离游离的dsDNA探针；
    (3)向步骤(2)中分离的游离dsDNA探针中加入CRISPR/Cas12a蛋白和向导RNA，并检测所产生的光信号；以及
    (4)基于所产生的信号，对所述小分子的存在或含量进行检测。
  35. 如权利要求33或34所述的方法，其中，在步骤(1)中，所述别构转录因子和所述激活dsDNA的摩尔比为≥5:1；或者，所述别构转录因子和所述dsDNA探针的摩尔比为≥5:1。
  36. 如权利要求33或34所述的方法，其中，在步骤(1)中，先将所述别构转录因子与所述激活dsDNA混合形成复合体，然后移除未结合的激活DNA，再加入所述待测样品；或者，先将所述别构转录因子与所述dsDNA探针混合形成复合体，然后移除未结合的是所述dsDNA探针，再加入所述待测样品。
  37. 如权利要求30-36中任一项所述的方法，其中，在步骤(1)中，所述混合在室温下进行1min以上。
  38. 如权利要求30-37中任一项所述的方法，其中，在步骤(1)中，所述别构转录因子固定在介质上。
  39. 如权利要求38所述的方法，其中，在步骤(1)之前，加入介质使所述别构转录因子固定至介质上。
  40. 如权利要求30-39中任一项所述的方法，其中，所述待测样品来自环境、受试者、食品、饮用水、化妆品或饲料。
  41. 如权利要求40所述的方法，其中，所述待测样品选自以下中的一种或多种：地下水、中水、海水、废水、采矿废料；血液、血清、血浆、痰、脑脊液流体、尿液、泪液、肺泡分离物、胸膜液、囊液、组织、唾液。
  42. 如权利要求30-41所述的方法，其中，在步骤(1)之前，对所述待测样品进行富集、提取和/或纯化的预处理。
  43. 如权利要求30-42中任一项所述的方法，其中，在步骤(2)中，通过过滤、离心、沉降、磁珠法、层析法、亲和柱法进行分离所述游离的dsDNA。
  44. 如权利要求30-43中任一项所述的方法，其中，在步骤(3)中，通过荧光分析或比色分析进行检测。
  45. 如权利要求40-44中任一项所述的方法，其中，在步骤(4)中，基于参比水平，对所述光信号进行分析，所述参比水平为所测定的不含所述小分子的样品的水平或含有已知量的小分子的样品的水平。
  46. 权利要求1-26中任一项所述的组合物在制备检测小分子的试剂盒中的用途。
  47. 如权利要求46所述的用途，其中，所述试剂盒用于环境污染监控、食品和化妆品质量控制和疾病诊断。
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