CN111812066A

CN111812066A - Biosensor based on CRISPR/Cas12a system, kit and application of biosensor in small molecule detection

Info

Publication number: CN111812066A
Application number: CN201910285607.1A
Authority: CN
Inventors: 张立新; 谭高翼; 梁敏东; 王为善; 刘家坤
Original assignee: East China University of Science and Technology; Institute of Microbiology of CAS
Current assignee: East China University of Science and Technology; Institute of Microbiology of CAS
Priority date: 2019-04-10
Filing date: 2019-04-10
Publication date: 2020-10-23
Anticipated expiration: 2039-04-10
Also published as: CN111812066B; WO2020207453A1

Abstract

The present invention relates to biosensors, compositions, kits comprising an allosteric transcription factor and a CRISPR/Cas12a system, and methods and uses related thereto. The mechanism of the biosensor is that an allosteric transcription factor and a CRISPR/Cas12a mediated small molecule detection tool are adopted, the binding of a target small molecule and the allosteric transcription factor changes the affinity of the allosteric transcription factor and double-stranded DNA, and a dsDNA probe and/or a ssDNA probe are/is cut by the cis-cutting activity and/or the trans-single-stranded DNA cutting activity of CRISPR/Cas12a, so that a target small molecule signal is converted into a corresponding optical signal. The biosensor, the composition, the kit and the related method can save time and cost when detecting small molecules in vitro, have high sensitivity and have the potential of high-throughput detection.

Description

Biosensor based on CRISPR/Cas12a system, kit and application of biosensor in small molecule detection

Technical Field

The invention belongs to the field of biological detection, and relates to a biosensor, a composition and a kit for converting a small molecule signal into an optical signal through cis-cleavage and trans-cleavage activities of CRISPR/Cas12a by means of an allosteric transcription factor (aTF) and a CRISPR/Cas12a system, and an in vitro (in vitro) detection method for the small molecule by using the biosensor, the composition and the kit.

Background

The detection of small molecules is so important, and is mainly applied in various fields such as clinical diagnosis, drug research and development, food safety, environmental monitoring and the like. For example, uric acid, which is an end product of purine metabolism, is generally in dynamic equilibrium between production and excretion in the body, and excessive accumulation affects normal functions of human cells. Among them, hyperuricemia, gout and nephropathy are all indicated by uric acid, are biomarkers in detection, and play an important role in the field of disease monitoring. Because of the inhibition effect of p-hydroxybenzoic acid (p-HBA) on fungi and bacteria, the p-hydroxybenzoic acid is widely applied to the fields of food preservatives, drug synthesis and cosmetics, and once exceeding standard, the harm is also huge. Therefore, the development of a new small molecule detection method is of great importance.

The method is extremely important for effectively detecting the micromolecules in the sample in the fields of environmental pollution monitoring, food quality control, disease diagnosis and the like. The detection methods of small molecules in the prior art are mainly divided into two categories: physical and chemical analysis method and biological analysis method. The physical and chemical analysis method mainly adopts a wave spectrum method, a chromatography method and a combination technology thereof. These techniques have high separation efficiency, good selectivity, and strong qualitative and quantitative abilities. However, the sample preparation process is complicated, the instrument is expensive, and the time is long. Even a skilled operator requires a relatively long time to obtain results and is not suitable for field analysis.

Bioanalytical methods typically rely on the specific interaction of small molecules with proteins/nucleic acids inherent in the body to achieve signal generation, amplification, signal conversion and readout on smaller systems or chips. The method can realize on-site and real-time detection. In particular, small molecules are captured using receptors or antibodies that interact with the small molecule, typically by mimicking agonist/antagonist-receptor, or antigen-antibody interaction pairs. Alternatively, specific recognition of small molecules is achieved by screening or evolving to obtain nucleic acids (e.g., aptamers) that specifically interact with small molecules. Then, signals of the interaction are converted and amplified into light/color development/electrochemical signals through an antibody cascade amplification mode, a quartz crystal microbalance mode and the like, and qualitative or quantitative information of the small molecules is read.

In order to expand the range of small molecules that can be analyzed, it has been proposed to convert information such as the presence or absence or concentration of small molecules into binding signals of the interaction of allosteric transcription factor-target DNA-small molecules by utilizing the interaction of allosteric transcription factors with their target DNA fragments and effector small molecules. The currently newly reported systems such as aTF-NAST (aTF-based normalized DNA-template-associated signal transmission) and the like further improve the sensitivity of uric acid and p-hydroxybenzoic acid, but the sample analysis period is long, the cost is high, and the rapid and batch detection is difficult to realize. Therefore, there is still a need in the art to establish a simple, rapid, highly sensitive, highly specific and suitable method for high-throughput in vitro detection of small molecule compounds.

The CRISPR/Cas system is an acquired immune defense mechanism evolved in the process that bacteria and archaea resist invasion of exogenous nucleic acid by using Cas protein under the guidance of RNA. A Clustered repetitive DNA sequence with unknown function is reported for the first time in 1978, and formally named Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) until 2001. With the continuous exploration of researchers in recent years, the classification, action mechanism and the like of the CRISPR system are preliminarily analyzed, and the CRISPR system is widely applied to various fields of gene editing, DNA assembly, gene expression regulation and the like.

According to the composition of Cas (CRISPR-associated proteins) genes and the quantity of effector proteins, CRISPR/Cas systems are divided into 2 types and 5 types; class 1 CRISPR/Cas systems interfere with target genes using multiple effector protein complexes; the class 2 CRISPR/Cas system utilizes a single effector protein to resist the invasion of exogenous nucleic acid, and is widely concerned due to simplicity and high efficiency. Among them, Cas9 and Cas12a (also referred to as Cpf1) have been successfully applied to the fields of prokaryotic/eukaryotic gene editing and gene regulation as typical of class 2 CRISPR/Cas systems. Based on the trans-cleavage activity of Cas12a, a diagnosis System of 'DETECTR' (DNAdende targeted CRISPR trans reporter) developed by Doudna team and 'HOLMES' (an one-HOur Low-cost Multipurpose high Efficient System) characterized by Zhao screen team in the recent past further promotes the application prospect of the CRISPR/Cas12a System in the field of nucleic acid detection. However, no report on the application of the CRISPR/Cas system in the detection of small molecule compounds exists at present.

Disclosure of Invention

In a first aspect, the present invention provides a biosensor for detecting small molecules, the biosensor comprising an identification element and a transducing element, wherein,

a: the recognition element comprises an allosteric transcription factor (aTF) and an activating double stranded DNA (activating dsDNA) comprising the binding site of aTF, the recognition site of the CRISPR/Cas12a system (i.e., protospacer adjacent motif, PAM) and a sequence that is at least partially complementary to the guide RNA, and

the transduction element comprises the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the guide RNA (gRNA), and both ends of the ssDNA probe are respectively conjugated with a luminescent/chromogenic group and a quenching group thereof;

or

B: the recognition element comprises an allosteric transcription factor (aTF) and a double-stranded DNA probe (dsDNA probe) comprising a binding site for the allosteric transcription factor, a recognition site PAM for the CRISPR/Cas12a system and a sequence at least partially complementary to a guide rna (grna), and both ends of the dsDNA probe are conjugated with a luminescent/chromogenic group and a quencher thereof, respectively, and

the transducing element comprises the CRISPR/Cas12a system, the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein with the gRNA.

In embodiments of the invention, upon contacting said biosensor with a small target molecule, binding of said aTF to the small target molecule increases or decreases the binding affinity of said allosteric transcription factor to said activated dsDNA and/or dsDNA probes, thereby decreasing or increasing the free form of activated dsDNA and/or dsDNA probes; the free form of the activated dsDNA forms a ternary complex with the CRISPR/Cas12a protein and the gRNA as an activator, thereby activating the trans-ssDNA cleavage activity of the CRISPR/Cas12a protein such that the ssDNA probe is cleaved and a detectable light signal is produced, or the free form of the dsDNA probe is recognized by the gRNA and cleaved by the CRISPR/Cas12a protein via cis-cleavage activity, thereby producing a detectable light signal; by means of the generated optical signal, the presence and/or the amount of the target microanalysis can be detected.

In a second aspect, the present invention provides a kit for detecting a small molecule, the kit comprising a biosensor as described in the first aspect. In some embodiments, the kit further comprises a detection element for detecting an optical signal.

In a third aspect, the present invention provides a method of detecting a small molecule in a sample, the method comprising: contacting the sample with the biosensor of the first aspect to generate an optical signal, and detecting the generated optical signal to detect the small molecule in the sample.

In a fourth aspect, the present invention provides a composition for detecting a small molecule, the composition comprising a recognition reagent and a transduction reagent, wherein,

a: the recognition reagent comprises an allosteric transcription factor (aTF) and an activating double stranded DNA (activating dsDNA) comprising the binding site of aTF, the recognition site PAM of the CRISPR/Cas12a system and a sequence at least partially complementary to the guide RNA, and

the transduction reagent comprises the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the guide RNA (gRNA), and both ends of the ssDNA probe are respectively conjugated with a luminescent/chromogenic group and a quenching group thereof;

or

B: the recognition reagent comprises aTF and a double-stranded DNA probe (dsDNA probe), the dsDNA probe comprises the binding site of aTF, a recognition site PAM of a CRISPR/Cas12a system and a sequence at least partially complementary to a guide RNA (gRNA), and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and a quencher thereof, and

the transduction reagent comprises a CRISPR/Cas12a system, and the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the gRNA.

In a fifth aspect, the present invention provides a kit for detecting a small molecule, the kit comprising the composition of the fifth aspect.

In a sixth aspect, the present invention provides a method for detecting a small molecule, the method comprising contacting a sample to be tested with the composition of the fifth aspect or the kit of the sixth aspect to generate an optical signal, and detecting the generated optical signal, thereby detecting the small molecule.

In a seventh aspect, 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 for detecting small molecules.

Advantageous effects

Compared with the traditional small molecule detection method, the invention adopts aTF and CRISPR/Cas12a mediated small molecule detection tool (CRISPR/Cas12a-andaTF-mediatedsmallmolecule detectorAbbreviated as "CaT-SMelor") by binding of the target small molecule to aTF resulting in a change in affinity of aTF to double stranded DNA and cis of CRISPR/Cas12aCleavage activity and trans-single stranded DNA cleavage activity, cleaving the dsDNA probes and/or ssDNA probes, converting the small molecule signal of interest into a corresponding optical signal, without involving aTF-target DNA binding DNA transcription regulation processes. The detection method provided by the invention is simple and convenient to operate, saves time (at most 25 minutes, even only 5-10 minutes) and cost (each reaction only needs less than 2 yuan), has high sensitivity (the detection limit is reduced to nM level), high accuracy and small reaction system (for example, a blood sample only needs 1 mu L), can be operated on a 96-well plate or a 384-well plate, and is suitable for high-flux accurate detection for diagnostic purposes and non-diagnostic purposes.

Drawings

FIG. 1 is a schematic diagram of the principle of small molecule detection using the biosensor of the present invention, according to one embodiment of the present invention. In this embodiment, aTF was immobilized for the convenience of subsequent operations and for the improvement of detection sensitivity. Specifically, aTF was expressed as a fusion with cellulose domain (CBD), and then the fusion protein aTF-CBD was mixed with Microcrystalline Cellulose (MC) to immobilize it on the MC; fusion protein aTF-CBD binds to activating dsDNA to form aTF-CBD-DNA complex; binding of the small molecule to aTF results in a aTF allosteric effect that causes the activated dsDNA to detach from aTF, resulting in a free form of activated dsDNA (also referred to as "free dsDNA"); adding free form of activated dsDNA to the CRISPR/Cas12a system and ssDNA probe (reporter: FQ-labeled ssDNA), which activates the CRISPR/Cas12a system and causes it to cleave the probe in trans, resulting in the emission of an optical signal (fluorescent signal); the optical signal is then detected. According to the embodiment of the present invention, the amount of the blood sample to be used is small (1. mu.L), and the reaction system has a small volume, and can be operated on an 96/384 well plate. And detecting optical signals by using common instruments such as a microplate reader and the like, thereby realizing qualitative/quantitative analysis of small molecules.

FIG. 2 is a graph illustrating the binding ability of the fusion proteins CBD-aTF (CBD-HucR, CBD-HosA and CBD-TetR) prepared in examples 1-2 to dsDNA by gel blocking assay (EMSA), and the dissociation of CBD-aTF-dsDNA in the presence of specific small molecules, according to an embodiment of the present invention. FIG. 2A:schematic structural diagrams of the fusion proteins CBD-HosA, CBD-HucR and CBD-TetR. The allosteric transcription factors HucR, HosA and TetR are connected with the CBD through a linker (the amino acid sequence of the linker can be shown as SEQ ID NO: 12); for ease of purification and identification, the fusion protein also has a His tag. Fig. 2B-fig. 2C: shows the fusion protein CBD-HucR with activating dsDNA (dsDNA) in the absence or presence of uric acid_(HucR)) Combined plot of (measured with EMSA). Fig. 2D-fig. 2E: shows the fusion protein CBD-TetR with the activating dsDNA (dsDNA) in the absence or presence of tetracycline_(TetR)) Combined plot of (measured with EMSA). Fig. 2F-fig. 2G: shows that the fusion protein CBD-HosA is bound to the activating dsDNA (dsDNA) in the absence or presence of p-hydroxybenzoic acid (p-HBA)_(HosA) Graphs of binding (measured using EMSA).

FIG. 3 is a graph showing the detection of a target small molecule using the biosensors of examples 1-2 according to one embodiment of the present invention. FIG. 3A: a flow chart for detecting small molecules using the biosensors of examples 1-2 of the present invention is shown. FIG. 3B: shows the fusion protein CBD-HucR with activating dsDNA (dsDNA) in the presence of 500. mu.M uric acid and its structural analogs (adenine, guanine, hypoxanthine)_(HucR)) Combined plot of (measured with EMSA). FIG. 3C: shows a graph of the fluorescence intensity detected using the uric acid biosensor of example 1 of the present invention with 500. mu.M Uric Acid (UA) and its structural analogs (adenine, guanine, hypoxanthine) as samples, in which free activated dsDNA (dsDNA) is present at 40nM_(HucR)) As a Positive Control (PC). FIG. 3D: shows that the fusion protein CBD-HosA and activating dsDNA (dsDNA) in the presence of 1.8mM p-hydroxybenzoic acid (p-HBA) and its structural analogue (Tyrosol (also known as "Tyrosol"), p-aminobenzoic acid (p-ABA), methylparaben (p-MHB), p-hydroxybenzyl alcohol (p-HBnOH))_(HosA) Graphs of binding (measured using EMSA). FIG. 3E: a graph showing fluorescence intensities detected using the p-HBA biosensor of example 2 of the present invention with 1.8mM p-HBA and its structural analogs (Tyrosol, p-ABA, p-MHB, p-HBnOH) as a sample, wherein,free activated dsDNA at 40nM (dsDNA)_(HosA) As a Positive Control (PC).

Fig. 4 is a graph showing detection of a target small molecule using the biosensors of examples 1 to 2 according to the present invention, according to an embodiment of the present invention. FIG. 4A: and (3) detecting the influence of the activated dsDNA with different concentrations on the change of fluorescence intensity, and taking the slope of a fluorescence value linear increasing interval to calculate a regression equation representing the linear detection range of the activated dsDNA by taking the concentration of the activated dsDNA as an abscissa and the corresponding slope as an ordinate. FIG. 4B: and (3) detecting the influence of uric acid with different concentrations on the change of fluorescence intensity, taking the slope of a fluorescence value linear increasing interval, and calculating the linear detection range of uric acid to be 25-500nM by taking the uric acid concentration as an abscissa and the corresponding slope as an ordinate. FIG. 4C: and (3) detecting the influence of the p-HBA with different concentrations on the change of the fluorescence intensity, taking the slope of a fluorescence value linear increasing interval, and calculating the linear detection range of the p-HBA to be 9-180nM by taking the p-HBA concentration as an abscissa and the corresponding slope as an ordinate.

Fig. 5 is a graph showing the measurement of uric acid concentration in a human blood sample using the uric acid biosensor of example 1 of the present invention, as compared with conventional uric acid concentration measurement methods (HPLC and Backman kit), according to an embodiment of the present invention. FIG. 5A: a graph of uric acid detection of human blood samples using the uric acid biosensor of the present invention and an HPLC method is shown. FIG. 5B: shows a chart for detecting uric acid in a human blood sample by using the uric acid biosensor and a clinically used Backman kit. FIG. 5C: a flow chart for using the biosensor of the present invention in blood sample analysis is exemplarily illustrated.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

According to the invention, CaT-SMelor is adopted, through the combination of the target small molecule and aTF, the affinity change of aTF and double-stranded DNA, the cis-form cutting activity and the trans-form single-stranded DNA cutting activity of CRISPR/Cas12a are used for cutting the dsDNA probe and/or the ssDNA probe, the target small molecule signal is converted into a corresponding optical signal, and the qualitative detection of the small molecule is realized. Further, the present inventors have found that for a small target assay, the resulting change in the slope of the fluorescence curve is proportional to the concentration of the small molecule over a range of concentrations, and thus accurate quantitative detection of the small target molecule in a sample can be achieved by measuring the slope of the fluorescence curve of a sample containing the small target molecule.

Composition comprising a metal oxide and a metal oxide

The 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) comprising the binding site of aTF, the recognition site PAM of the CRISPR/Cas12a system, and a sequence at least partially complementary to a guide RNA; and the transducing reagent comprises the CRISPR/Cas12a system,

wherein the composition further comprises a luminescent/chromogenic group.

In one embodiment, the transduction reagent further comprises single-stranded dna (ssdna).

In particular, the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the guide rna (grna).

In a specific embodiment of the invention, the dsDNA may serve as an activating dsDNA, or dsDNA probe. In another embodiment, the ssDNA may also be used as a probe. In the case of use as a probe, both ends of the dsDNA probe or ssDNA probe may be conjugated with a luminescent/chromogenic group and a quencher thereof.

In one embodiment, the composition of the invention comprises a recognition reagent comprising an allosteric transcription factor (aTF) and an activating double stranded DNA (activating dsDNA) comprising the binding site of aTF, the recognition site PAM of the CRISPR/Cas12a system and a sequence at least partially complementary to a guide RNA, and a transduction reagent

The transduction reagent comprises the CRISPR/Cas12a system and a single-stranded DNA probe (ssDNA probe), the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the guide RNA (gRNA), and both ends of the ssDNA probe are respectively conjugated with a luminescent/chromogenic group and a quenching group thereof.

In another embodiment, the composition of the invention comprises a recognition reagent comprising aTF and a double-stranded DNA probe (dsDNA probe) comprising the binding site of aTF, the recognition site PAM of the CRISPR/Cas12a system, and a sequence at least partially complementary to a guide rna (grna), and a light/color-producing group and a quencher thereof conjugated to both ends of the dsDNA probe, respectively, and a transduction reagent, and

In an embodiment of the invention, the composition of the invention may be in the form of a biosensor.

Biosensor and method for measuring the same

Biosensors (biosensors), generally known as a device for analyzing biological materials (e.g., tissues, microbial cells, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.) or biologically derived or biomimetic materials, can closely associate or associate a biological material or the like with a physicochemical sensor or a sensing microsystem (which may be optical, electrochemical, thermal, piezoelectric, or magnetic) and can produce an intermittent or continuous signal (optical, electrical, etc.) whose intensity is proportional (e.g., linear) to the analyte to perform an analytical function. Biosensors have a general definition in the art, and are generally composed of two parts: a recognition element (also known as a molecular recognition element) and a transduction element (also known as a transducer). In this context, the term "biosensor" refers to a device or apparatus comprising an identification element and a transduction element, also referred to as a "biosensing platform". Without being bound by theory, the biosensors described herein may include amplification elements that further increase detection sensitivity and/or detection elements that directly perform detection. Furthermore, the biosensor described herein is not limited to a particular form, as long as any form including the identification element and the transduction element described below is included within the scope of the term "biosensor" herein.

Identification agent or element

The recognition element is a key element of the biosensor, which recognizes the analyte, and performs a biological reaction, directly determining the function and quality of the sensor. Herein, the recognition element comprises aTF and activating dsDNA, or aTF and a dsDNA probe.

The term "allosteric transcription factor" as used herein has the meaning well known in the art. To accommodate changes in the external environment and respond in time, microbes have evolved allosteric protein aTF as an important input element for sensing the environment, aTF typically comprises a DNA Binding Domain (DBD) and an Effector Binding Domain (EBD), wherein effector binding alters aTF binding to target gene sequences, thereby regulating expression of the associated genes and responding rapidly to external changes. According to aTF sequence similarity and difference of structure and function, the compounds can be divided into different families such as AraC, LacI, TetR and the like, and these aTF can specifically sense various small molecule compounds and play an important role in maintaining metabolic stability.

An allosteric transcription factor comprises an effector (usually a small molecule) binding domain (EBD) and a DNA Binding Domain (DBD), the binding of which to the effector causes a conformational change that alters the binding affinity of the allosteric transcription factor to a DNA fragment (usually a promoter operator in nature) that specifically interacts with it, thereby enhancing or reducing transcription of a DNA sequence controlled by the operator (Nat methods.2016, 13 (2): 177-183) in such a way that transcription of the gene is dependent on the concentration of small molecules. In prokaryotes, the operator sequence is usually present upstream of a metabolically relevant operator or reporter gene; allosteric transcription factors often play a role of sensors for effector small molecules in cells, and feed back the concentration information of the small molecules, so that the biosynthesis pathway in the cells is dynamically regulated. In eukaryotes, such allosteric transcription factors are often present in pathways that control cell differentiation and ontogeny. As the interaction of the allosteric transcription factor with DNA in the present invention occurs in vitro, allosteric transcription factors of both prokaryotic and eukaryotic origin can be used as recognition elements in the present invention. In the present invention, the transcription factor binding site is also referred to as a transcription factor action site, a transcription factor operation site, or a transcription factor binding motif. Without wishing to be bound by theory, the transcription factor site of action is typically complementary double stranded DNA. Furthermore, it will be appreciated by those skilled in the art that the presence of a single 3',5' -phosphodiester bond break (nick) near the site of action of a transcription factor does not affect the binding of the allosteric transcription factor to its site of action.

The sequence of the site of action of transcription factors in natural transcription systems is known. The transcription factor binding sites of different species vary slightly in length. The average length of the transcription factor binding site in E.coli is 24.5bp and in Drosophila 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 10-40bp, more preferably 15-25bp, most preferably 17-19 bp. The DNA fragment which acts with the allosteric transcription factor is not limited to the allosteric transcription factor action site which exists in the natural system. It is well known in the art to select several candidate sequences by random or directed mutagenesis of the site of action sequence, or in silico, and to verify the binding ability of allosteric transcription factors to each DNA fragment (Mol Microbiol.2005, 55 (3): 712-23.). In addition, mutations can be made to the small molecule binding domain and/or the DNA binding domain of the allosteric transcription factor to optimize the binding affinity of the allosteric transcription factor to the small molecule and/or DNA. The sensitivity of the sensor can be further improved by improving the equilibrium dissociation constant between the allosteric transcription factor and the DNA fragment acting with the allosteric transcription factor and/or reducing the equilibrium dissociation constant between the allosteric transcription factor and the small molecule through directed mutation and evolution.

As described above, binding of an allosteric transcription factor to a small molecule can result in either an increase (activation system) or a decrease (repression system) in the binding affinity of the allosteric transcription factor to a target dsDNA fragment (i.e., a DNA fragment comprising the site of action of the allosteric transcription factor, also referred to herein as an "activated dsDNA" or "dsDNA probe"). In some embodiments, an allosteric transcription factor of the invention is an allosteric transcription factor that activates a system. In this system, binding of the allosteric transcription factor to the effector small molecule results in an increased binding affinity to the target dsDNA (i.e., binding of the small molecule enables the allosteric transcription factor to bind to the target dsDNA fragment), thereby reducing the amount of free target dsDNA (i.e., target dsDNA not bound to the allosteric transcription factor). Preferably, the equilibrium dissociation constant for binding of the transcription factor-small molecule complex formed upon binding of the allosteric transcription factor to the small molecule to the target dsDNA fragment is greater than the equilibrium dissociation constant for binding of the allosteric transcription factor to the target dsDNA fragment; preferably, the equilibrium dissociation constant for binding of the allosteric transcription factor-small molecule complex to the target dsDNA fragment is 10-10000 times, preferably 20-5000 times, more preferably 50-1000 times the equilibrium dissociation constant for binding of the allosteric transcription factor to the target dsDNA fragment. In some embodiments, an allosteric transcription factor of the invention is an allosteric transcription factor of a repression system. In this system, binding of the allosteric transcription factor to the effector small molecule results in a decrease in its binding affinity to the target dsDNA (i.e., binding of the small molecule results in detachment of the allosteric transcription factor from its site of action, increasing the amount of free target dsDNA). Preferably, the equilibrium dissociation constant for binding of the allosteric transcription factor to the small molecule is greater than the equilibrium dissociation constant for binding of the allosteric transcription factor to the target dsDNA fragment; preferably, the equilibrium dissociation constant for binding of the allosteric transcription factor to the small molecule is 10-10000 times, preferably 20-5000 times, more preferably 50-1000 times the equilibrium dissociation constant for binding of the allosteric transcription factor to the allosteric transcription factor of the invention to the target dsDNA fragment. In some embodiments, the allosteric transcription factor and target dsDNA are provided as a composition or combined complex. In other embodiments, the allosteric transcription factor and the target dsDNA are provided as separate compounds. It should be particularly noted that although only the allosteric transcription factor of the repressor system is used in the specific embodiments of the invention, this is merely illustrative, and based on the teachings of the present application, one skilled in the art can practice the invention using different allosteric transcription factors (e.g., other allosteric transcription factors of the repressor system or allosteric transcription factors of the activator system) as needed.

The biosensors, compositions, kits and methods of the invention can be used for diagnostic or non-diagnostic use, or non-clinical use, for example for environmental pollution monitoring, food and cosmetic quality control and disease diagnosis. In the present invention, the small molecule to be detected is an effector that causes conformational change of an allosteric transcription factor. In general, a small molecule is characterized in that it has a molecular weight greater than about 50 daltons but less than about 5000 daltons (5 kD). Preferably the small molecule has a molecular weight of less than 1 kD. In the present invention, the small molecule may be, for example, an environmental indicator, a disease indicator, or a health indicator, including but not limited to, heavy metal ions, toxins, drugs, metabolites, pollutants, or decomposition products of the foregoing, and the like. Small molecules may be present in the environment or of bacterial, fungal, plant or animal origin, or be artificially synthesized. Even for effector small molecules where no corresponding allosteric transcription factor is present, one skilled in the art can construct artificial allosteric transcription factors by in silico design by adding effector binding domains to DNA binding domains, or engineering the effector binding domains of natural transcription factors (natmethods.2016, 13 (2): 177-.

In the present invention, the small molecule to be detected may be present in any liquid sample or in a solid sample that can be converted into a liquid sample by suitable manipulation. The sample may be an environmental sample, such as a sample of groundwater, reclaimed water, seawater, wastewater, mining waste. Alternatively, the sample may be a biological sample, in particular a sample from a subject, for example one or more of the following: blood, serum, plasma, sputum, cerebrospinal fluid, urine, tears, alveolar isolates, pleural fluid, cystic fluid, tissue, saliva. The sample may also be derived from food, drinking water, cosmetics or feed.

In some embodiments, the sample can be pretreated, so as to detect the small molecules are enriched and extracted, or to remove may interfere with the detection of impurities. For example, the pretreatment may be performed by centrifugation, filtration, sonication, homogenization, heating, freezing, thawing, mechanical treatment, or a combination of various manipulation methods, and/or the addition of pretreatment reagents. The person skilled in the art is aware of the usual pretreatment methods and pretreatment reagents for a particular sample. For example, commonly used pretreatment reagents include surfactants and detergents, salts, cell lysing agents, anticoagulants, degradative enzymes (e.g., proteases, lipases, nucleases, lipases, collagenases, cellulases, amylases, etc.), and solutions (e.g., buffers), and the like.

Table 1 lists a number of allosteric transcription factors, their interacting DNA sequences and the corresponding effector small molecules. It will be appreciated by those skilled in the art that the allosteric transcription factors for use in the present invention are not limited to those listed. In addition, one or more bases of the DNA sequence interacting with the enumerated allosteric transcription factors may be substituted, deleted, or added to change the binding strength between the allosteric transcription factors and the DNA sequence interacting therewith.

Table 1: exemplary allosteric transcription factors, DNA sequences interacting therewith, and corresponding effectors

In some embodiments of the invention, the recognition element comprises 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 comprises an allosteric transcription factor that can be immobilized on a medium and an activating dsDNA or dsDNA probe that can be immobilized on a medium. The term "media" refers to a substance that supports and provides a reaction space for each element, thereby facilitating carrying, transporting, packaging, or handling. The element to be fixed is usually fixed on the medium by immobilization techniques such as adsorption, covalent bonding, physical embedding and cross-linking. Examples of media include, but are not limited to, nitrocellulose or nylon membranes, affinity column chromatography matrices, magnetic beads, solid fillers, microcrystalline cellulose, or commercially available protein immobilization media, and the like. In a preferred embodiment, the medium is microcrystalline cellulose. In some preferred embodiments, the recognition element of the present invention comprises an allosteric transcription factor that can be immobilized on microcrystalline cellulose. In a further preferred embodiment, the allosteric transcription factor fixable to microcrystalline cellulose is an allosteric transcription factor expressed in fusion with a cellulose domain (hereinafter referred to as "CBD"), and the fusion protein is fixable to microcrystalline cellulose by adsorption between the CBD and microcrystalline cellulose.

In some embodiments of the invention, the recognition element comprises an allosteric transcription factor expressed with a CBD fusion protein. As shown in the specific examples, the present inventors expressed the cellulose domain fused with an allosteric transcription factor to obtain a fusion protein that can be immobilized on microcrystalline cellulose. For example, a schematic of the structure of such fusion proteins is shown in FIG. 2A, where an allosteric transcription factor (e.g., HucR, HosA, TetR, whose amino acid sequences can be shown in SEQ ID NOs:1-3, respectively) is joined to a CBD (whose amino acid sequence can be shown in SEQ ID NO: 4) with a linker. The purpose of the linker is simply to link aTF to the CBD, and the amino acid sequence can be as shown in FIG. 2A (SEQ ID NO:12), but is not limited thereto, and one skilled in the art can select other linkers according to actual needs. The present inventors experimentally confirmed that the structural transcription factors expressed as fusion proteins (CBD-HucR, CBD-HosA and CBD-TetR) did not adversely affect their binding ability to activating dsDNA and allosteric activity (as shown in FIGS. 2B-2G). Furthermore, the DNA binding ability and allosteric activity of the fusion protein were compared with that of the separately expressed allosteric transcription factor, and no significant difference was found between the two. This indicates that the CBD only serves the purpose of immobilizing the allosteric transcription factor to microcrystalline cellulose, and does not affect the function of the allosteric transcription factor. Methods for expressing and purifying allosteric transcription factors fused to CBDs are well known in the art. For example, a gene encoding an allosteric transcription factor having an amino acid sequence represented by SEQ ID NOs:1-3 and a gene encoding a cellulose domain having an amino acid sequence represented by SEQ ID NO:4 are cloned into an expression vector (e.g., a His-tagged pET23b vector), thereby constructing a pET23b-aTF-CBD plasmid; the plasmid is transferred into an expression bacterium (escherichia coli BL21), and the fusion protein can be obtained through induced expression. Further, the purification can be carried out by a protein purification method generally used in the art, such as affinity column chromatography, fast protein liquid chromatography, and the like. In the present invention, the fusion protein comprises a purified fusion protein. Without being bound by a particular theory, other elements of the present invention (e.g., the transducer or detector elements described below) may also be immobilized on the media, and modifications or improvements may be made by one skilled in the art according to actual needs, and are also encompassed within the scope of the present invention.

The term "activating dsDNA" or "activating dsDNA fragment" as used herein is a nucleotide comprising a DNA fragment of the allosteric transcription factor action site, the CRISPR/Cas12a protein recognition site PAM and a sequence at least partially complementary to the gRNA, which 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 activating dsDNA can act as an activator to activate the trans-cleavage activity of the CRISPR/Cas12a system and is therefore referred to herein as "activating dsDNA" and as mediating the initiation of the transducing element, can also be referred to as "transducing DNA". In the present invention, the length of the activating dsDNA is preferably 20-80bp, more preferably 55-65bp, most preferably 58-60 bp. Wherein the definition of allosteric transcription factor binding site is as described above.

The CRISPR/Cas12a protein recognition site PAM as described herein refers to a sequence that is recognized by the CRISPR/Cas12a system and thus activates the cleavage activity of the CRISPR/Cas12a system, which is referred to as a Protospacer Adjacent Motif (PAM), generally T-rich (for CRISPR/Cas12 a).

In the present invention, the length of PAM is preferably 3-8bp, more preferably 3-6bp, most preferably 4 bp. The PAM sequence is not limited to PAM that is present in the natural system. The selection of several candidate sequences by random or directed mutagenesis of the site of action sequence, or in conjunction with computer modeling, and validation of the ability of PAM to bind to CRISPR/Cas12a is a technique known in the art. For example, in an embodiment of the invention, the nucleotide sequence that activates the allosteric transcription factor site of action in dsDNA and the PAM sequence are as follows (SEQ ID NOS: 5-7): tactgagccatgtatccaggtcattgTACTTAGATGTCTACCTAagctctgacagttcca, tactgagccatgtatccaggtcattgCGTTCGTATACGAACAgtagctctgacagttcca, tgagccatgtatccaggtcatttgTCCCTATCAGTGATAGAGAagctctgacagttcca (wherein the underlined portion is PAM and the bold portion is the allosteric transcription factor binding site, i.e., the portion complementary to the gRNA).

In the present invention, said "at least partially complementary" to a gRNA refers to a sequence that is at least 50% or more complementary, such as at least 70% or more complementary, for example 80%, 85%, 90%, 95%, 99% or more, or 100% complementary to said gRNA.

In embodiments of the invention, the sequence at least partially complementary to the gRNA may precede, follow, or be between the transcription factor binding site and the PAM, or at least partially overlap the transcription factor binding site and/or the PAM, or be located within the allosteric transcription factor binding site in activating the dsDNA. For example, in activating dsDNA, an allosteric transcription factor binding site is said sequence that is at least partially complementary to a gRNA. Thus, in a preferred embodiment, only PAM and allosteric transcription factor binding sites (which contain sequences complementary to the gRNA) may be included in the activating dsDNA. Thus, the length of the activating dsDNA can be as low as 13 or 14 bp.

In the present invention, in "activating dsDNA", the allosteric transcription factor action site may be separated from the PAM sequence by several nucleotides, for example, by 0-20bp, more preferably 5-10bp, most preferably 6-9 bp. In one embodiment, the allosteric transcription factor action site may be directly linked to the PAM sequence, or may partially or completely overlap. Alternatively, the PAM sequence may be located in the allosteric transcription factor site of action.

Herein, the "activating dsDNA" has both an allosteric transcription factor action site and a CRISPR/Cas12a protein recognition site PAM present thereon, and thus can bind to both an allosteric transcription factor and a Cas12a protein. The equilibrium dissociation constant for binding of the allosteric transcription factor to the activating dsDNA fragment can be higher, lower or similar to the equilibrium dissociation constant for binding of the CRISPR/Cas12a protein to the activating dsDNA fragment. In some embodiments of the invention, the equilibrium dissociation constant for binding of the allosteric transcription factor to the activating dsDNA fragment is lower than the equilibrium dissociation constant for binding of the CRISPR/Cas12a protein to the activating dsDNA fragment, i.e., the activating DNA fragment binds to the CRISPR/Cas12a protein more readily than the allosteric transcription factor. Therefore, in order to reduce interference of the allosteric transcription factor and the CRISPR/Cas12a system on each other's functions, reduce noise, and improve 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. In some embodiments, the activating dsDNA can be prepared by: for example, double-stranded DNA is formed by annealing (e.g., pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, reduction of 1 ℃ per cycle, 70 cycles; storage at 25 ℃) using a primer pair (e.g., the nucleotide sequences shown as SEQ ID NOs:5-18, 6-19, or 7-20) targeting a specific allosteric transcription factor action site and CRISPR/Cas protein recognition site; alternatively, it is also possible to artificially synthesize, for example, a double-stranded DNA having a nucleotide sequence shown in SEQ ID NOs: 5-7. The activated dsDNA can also be prepared by other means known in the art.

In one embodiment, to avoid aTF competing for binding to the CRISPR/Cas12a protein in the same reaction system to activate dsDNA, the dsDNA can be activated by fusion expression of aTF with other proteins that do not affect its activity (e.g., CBD), or such that there is overlap of allosteric transcription factor action sites with the PAM sequence in the activated dsDNA.

"probe" or "DNA probe" as used interchangeably herein refers to a nucleotide sequence that is complementary to a gRNA and is cleaved by the CRISPR/Cas12a system and has a group (also referred to herein as a "label") that, upon cleavage, can generate a detectable light signal. In some embodiments, the probe has a double-stranded region containing an allosteric transcription factor action site, a recognition site PAM of the CRISPR/Cas12a system, and a specific nucleotide sequence recognizable by the gRNA and/or crRNA, i.e., a sequence at least complementary to the gRNA and/or crRNA, or is a double-stranded DNA probe (both referred to as a dsDNA probe). In one embodiment, the dsDNA probe is, for example, 13-100bp, more preferably 20-50bp, most preferably 20-30bp in length. Wherein the definition of allosteric transcription factor binding site is as described above. In particular embodiments, the nucleotide sequence portion of the dsDNA probe may be identical to the activating dsDNA. In the present invention, the length of the sequence that can be recognized by the gRNA and/or crRNA is preferably 15 to 70bp, more preferably 15 to 30bp, and most preferably 17 to 24 bp. In a preferred embodiment, the dsDNA probe comprises a sequence that is at least 70% or more complementary, e.g., 80%, 85%, 90%, 95%, 99% or more, or 100% complementary to the gRNA and/or crRNA.

In the present invention, in the "dsDNA probe", the allosteric transcription factor action site may be separated from the sequence recognized by the gRNA and/or crRNA by several nucleotides, for example, by 0-20bp, more preferably 5-10bp, most preferably 6-9 bp. In one embodiment, the allosteric transcription factor action site may be directly linked to a sequence recognized by the gRNA and/or crRNA, or may partially or completely overlap. Alternatively, sequences recognized by the gRNA and/or crRNA may be located in the allosteric transcription factor site of action.

In some embodiments, the probe is a single-stranded DNA, which may be any nucleotide sequence, such as a nucleotide sequence of about 10-30bp, preferably about 20bp in length, as shown in SEQ ID NO. 8. Without being limited by a particular theory, in the case where the activating DNA of the present invention is a double-stranded DNA and can be designed to be recognized by a gRNA and/or crRNA, it is within the scope of the present invention for one skilled in the art to design the activating dsDNA of the present invention as a double-stranded DNA probe (e.g., fluorescently labeled at both ends of the activating DNA) to practice the present invention in light of the intent and principles of the present invention.

Transducing reagents or transducing elements

Herein, the "CRISPR/Cas system" refers to a class 2 CRISPR/Cas system, which uses a single effector protein to resist invasion of foreign nucleic acid, and enables simple and efficient gene editing and nucleic acid detection. Among them, the most typical class 2 CRISPR/Cas system is the CRISPR/Cas12a (Cpf1) system. Herein, a CRISPR/Cas system typically comprises at least a CRISPR/Cas protein and a guide RNA. CRISPR (clustered regularly interspaced short palindromic repeats) is a fragment of prokaryotic DNA containing repeats of a short base sequence. Each repetition is followed by a short fragment of "spacer DNA" from a previous exposure to a bacterial virus or plasmid. Cas (CRISPR-associated protein) is an RNA (guide RNA) -guided DNA endonuclease. Guide RNAs (grnas or sgrnas) are specific RNA sequences that guide recognition and cleavage of a target nucleic acid molecule by a CRISPR/Cas protein, and can be synthesized by in vitro transcription or artificial chemistry. The guide RNA may be formed by hybridization of CRISPR RNA (crRNA) and transactivating crRNA (tracrrna), or may be provided as a separate continuous RNA. The gRNA specifically binds to a complementary target sequence via a target-specific sequence (e.g., a spacer sequence "spacer") in the crRNA portion, while the CRISPR/Cas protein itself binds to the PAM, which then the Cas nuclease mediates cleavage of the target nucleic acid (e.g., DNA probe). However, for CRISPR/Cas12a, only crRNA guidance is required, and tracrRNA is not required; and CRISPR/Cas12a cleaves not only double strand DNA of a specific sequence (dsDNA, which contains a sequence complementary to the crRNA that is recognized by the crRNA) but also any single strand DNA (ssdna) when activated by the double strand DNA of a specific sequence (such as the activating DNA herein) to form a CRISPR/Cas/dsDNA ternary complex. These properties enable CRISPR/Cas12a to improve the sensitivity, specificity and speed of detection. Thus, in some embodiments, the CRISPR/Cas12a system is preferred. To the best of the inventor's knowledge, Cas12a itself cannot be used for detecting small molecule compounds in various fields such as disease diagnosis, drug development, food safety, and environmental monitoring. In the invention, the inventors can realize the detection of small molecule compounds by means of a recognition element (allosteric transcription factor-activated dsDNA complex) and when PAM (polyacrylamide gel) binding CRISPR/Cas protein on the dsDNA is activated to activate CRISPR/Cas to cut a probe (target nucleic acid) so as to generate a detectable optical signal.

In the present invention, the length of the gRNA is preferably 20-70bp, more preferably 30-50bp, most preferably 38-45 bp. grnas can be designed based on the needs of the user. The design is preferably performed in conjunction with bioinformatics software. In some embodiments, the light signal comprises a fluorescent signal or an absorbed light signal. It will be appreciated by those skilled in the art that in order to quantify the color change that occurs during analysis, the intensity of the absorbed light is typically detected after excitation with light of a particular wavelength. Thus, in the present invention, the term "light absorption signal" may also refer to the resulting color change (colorimetric analysis). In some embodiments, the probe can carry a label to generate a detectable light signal upon cleavage by CRISPR/Cas. Examples of markers include, but are not limited to: luminescent organic compounds (e.g., fluorescein, raphanin), luminescent inorganic compounds (e.g., chemical dyes), fluorophores (e.g., FAM fluorophores), and the like; nanoparticles, quantum dots, and the like; or chromophores, and the like. The technique of labeling nucleic acid sequences with the above substances to generate a detectable light signal is well known to those skilled in the art, and can be selected and modified according to practical needs, which does not limit the present invention. In some preferred embodiments, the optical signal is a fluorescent signal. In a further preferred embodiment, the labels are a luminophore and a quencher, respectively, at both ends (3 'end or 5' end) of the probe. Preferably, the luminophore is a FAM fluorophore and the quencher is a BHQ fluorescence quencher. In other preferred embodiments, the optical signal is an absorption optical signal. In a further preferred embodiment, the labels are a chromophore and a quencher, respectively, at both ends (3 'or 5' ends) of the probe.

Detection element

In the present invention, the detection of the optical signal using various detection devices is well known to those skilled in the art.

Detection method

In an embodiment of the invention, the detection of small molecules in a sample is performed using a biosensor according to the invention or a composition according to the invention. In the present invention, the small molecules in the sample can be detected qualitatively or quantitatively.

The invention provides a method for detecting small molecules in a sample to be detected, which comprises the following steps:

mixing and incubating a sample to be detected and an identification reagent; isolating free dsDNA fragments from the resulting mixture; adding the separated free dsDNA to a transduction reagent and detecting the resulting optical signal; and detecting the presence or amount of the small molecule based on the generated signal.

In one embodiment, the present invention provides a method for detecting a small molecule in a test sample, the method comprising:

(1) mixing and incubating a sample to be tested, aTF and dsDNA; (2) isolating free dsDNA fragments from the resulting mixture; (3) adding the isolated free dsDNA to the CRISPR/Cas12a protein and guide RNA and detecting the resulting optical signal; and (4) detecting the presence or amount of the small molecule based on the generated signal.

In one aspect, the present invention provides a method for detecting a small molecule in a test sample, the method comprising:

(1) mixing and incubating a sample to be tested, aTF and the activated dsDNA fragments;

(2) isolating free activated dsDNA fragments from the mixture in step (1);

(3) adding CRISPR/Cas12a protein, guide RNA and ssDNA probe to the free activated DNA fragment separated in step (2), and detecting the generated optical signal; and

(4) detecting the presence or amount of the small molecule based on the generated signal.

In another aspect, the present invention provides a method for detecting a small molecule in a test sample, the method comprising:

(1) mixing and incubating a sample to be tested, aTF and a dsDNA probe;

(2) isolating free dsDNA probes from the mixture in step (1);

(3) adding CRISPR/Cas12a protein and guide RNA to the isolated free dsDNA probe of step (2) and detecting the resulting optical signal; and

Wherein, the specific conditions for mixing and incubating in step (1) can be selected by those skilled in the art according to the actual needs, as long as it is ensured that the allosteric transcription factor and the activated dsDNA or dsDNA probe can be sufficiently combined into a complex and the specific small molecule compound in the sample to be tested can be sufficiently combined with the allosteric transcription factor-activated dsDNA or dsDNA probe complex, for example, reacting at room temperature for more than 1min, such as 15-20min (but not limited thereto). The ratio of allosteric transcription factors to activating dsDNA or dsDNA probes can be adjusted according to actual needs. In some embodiments of the invention, it is preferred that the molar ratio of allosteric transcription factor to activating dsDNA or dsDNA probes is greater than or equal to 5:1, in which case the allosteric transcription factor can fully bind to the activating dsDNA or dsDNA probes to form a protein-DNA complex.

In some embodiments, the allosteric transcription factor is an allosteric transcription factor of a repressor system (i.e., when a small molecule binds to the allosteric transcription factor, the binding affinity of the allosteric transcription factor to the target dsDNA is reduced, such that the target dsDNA is detached from the aTF-DNA complex), and the amount of free dsDNA increases upon addition of the test sample. Thus, in a preferred embodiment, to reduce background noise and increase the sensitivity of the present invention, allosteric transcription factors are first mixed with dsDNA to form aTF-dsDNA complexes, then unbound dsDNA is removed and the test sample is added. In this embodiment, since the allosteric transcription factor completely binds to dsDNA to aTF-dsDNA complex before the test sample is added, and there is substantially no free dsDNA, the dsDNA that is free after the test sample is added directly reflects the presence or amount of small molecules in the test sample.

In other embodiments, the allosteric transcription factor is an allosteric transcription factor that activates the system (i.e., when a small molecule binds to the allosteric transcription factor, the binding affinity of the allosteric transcription factor to dsDNA increases such that dsDNA binds to the allosteric transcription factor into more aTF-dsDNA complexes), and the amount of free dsDNA decreases upon addition of the test sample. It will be appreciated by those skilled in the art that in such embodiments, the amount of dsDNA added should be determined prior to step (1), and the reduced free dsDNA calculated indirectly reflects the presence or amount of small molecules in the sample to be tested. In some embodiments, the test sample can be added before, simultaneously with, or after mixing the dsDNA with the allosteric transcription factors.

In a preferred embodiment, the allosteric transcription factor is an allosteric transcription factor that can be immobilized on a medium. Thus, step (1) may further comprise adding a mediator, for example, microcrystalline cellulose when the allosteric transcription factor that can be immobilized on a mediator is an allosteric transcription factor expressed in fusion with a cellulose domain. Preferably, the allosteric transcription factor is pre-bound to the medium prior to addition of the test sample and dsDNA, and immobilized on the medium for subsequent manipulation. For example, a commercially available medium (e.g., microcrystalline cellulose) is mixed and incubated with an allosteric transcription factor that can be immobilized on the medium (e.g., an allosteric transcription factor expressed in fusion with a cellulose domain), and the specific conditions for mixing and incubation 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, for example, a reaction at room temperature for 5 to 15min (but not limited thereto). Preferably, the non-immobilized allosteric transcription factor is removed prior to addition of the test sample and dsDNA. Further, the amount or concentration of allosteric transcription factor immobilized on a medium can be determined, for example, using protein concentration detection methods known in the art (e.g., using a Bradford assay kit).

Methods for separating free dsDNA from the protein, small molecule compound and nucleic acid mixture of step (1) in step (2) are well known in the art and include, for example, but are not limited to, centrifugation, sedimentation, magnetic bead methods, chromatography, affinity column methods, and the like. The skilled person can select the specific separation method and parameters according to the actual situation, which is not intended to limit the present invention. In embodiments where the allosteric transcription factor is one that can be immobilized on a medium, the free dsDNA fragments can be isolated by filtration or centrifugation (e.g., 7000rpm at room temperature), which is simple to operate, time and cost saving, and economically viable. The separation step of step (2) can also be omitted and the CRISPR/Cas12a system and optional ssDNA probe added directly to the mixture of step (1).

In step (3), the optical signal generated is detected using methods and apparatus commonly used in the art for detecting fluorescent signals or absorbed optical signals. For example, when the signal generated is a fluorescent signal, it is measured using a microplate reader. It should be noted that when a qualitative analysis is performed on the sample or small molecule compound to be detected, the resulting color change (which, as mentioned above, is included in the "light absorption signal") may not be quantitatively analyzed, but only be calorimetrically analyzed, which is also within the scope of the claimed invention.

In step (4), the signal generated can be analyzed based on the reference level to detect the presence or amount of the small molecule. The "reference level" is used interchangeably herein with "reference sample" and "reference level" and refers to a control of conditions. For example, in the context of qualitative detection (detection of the presence or absence) of a small molecule, a reference level can be the level of a sample that does not contain the small molecule. In the context of quantitative detection (detection of amount) of small molecules, a reference level can be the level of a sample containing a known amount of small molecules. For determining the presence and amount of a sample comprising a small molecule, a reference level is a reference value that can be normalized to a suitable standard to infer the presence, absence, or amount of a small molecule in a sample. In some embodiments, the reference level may be a previously determined level, e.g., may be a predetermined quantity or ratio, and need not be determined in the same physical iteration of the detection method described herein.

In the present invention, the sample as well as the reagents may be in the form of a solution as long as aTF binding to effector/dsDNA and cleavage of CRISPR/Cas12a is not affected. And can be selected or adjusted by those skilled in the art according to actual application or requirements.

Reagent kit

The invention also provides a kit for detecting small molecules, which comprises the biosensor and the detection element. In this context, the detection element refers to an element, device or system that detects the optical signal generated by the biosensor of the present invention. In some embodiments, the detection element is used to analyze the optical signal generated by the DNA probe by fluorescence analysis or absorbance analysis (including colorimetric analysis) to obtain a qualitative or quantitative detection of the small molecule.

In another aspect, the invention also provides a kit for detecting small molecules, wherein the kit comprises the reagent and the detection reagent. In the present invention, the detection reagent is a reagent for detecting an optical signal generated by using the reagent of the present invention. In some embodiments, the detection reagent is used to analyze the optical signal generated by the DNA probe by fluorescence analysis or absorbance analysis (including colorimetric analysis) to obtain a qualitative or quantitative detection of the small molecule.

In some embodiments, a kit of the invention further comprises a delivery means or device (e.g., pipette) for detecting small molecules using the biosensors and/or reagents of the invention, a wash buffer, a dilution buffer, a stop buffer (e.g., for stopping color development), a microtiter plate (e.g., 98-well or 384-well for performing reactions and assays), one or more containers, a data carrier (e.g., instructions or computer readable medium) bearing instructions for use, a standard (e.g., a sample containing a known amount of small molecules), combinations thereof, and the like.

Embodiments of the aspects described herein may be illustrated by the following numbered paragraphs:

1. a composition for detecting a small molecule, the composition comprising a recognition reagent and a transduction reagent, wherein,

wherein the composition further comprises a luminescent/chromogenic group.

2. The composition of paragraph 1 wherein the transduction reagent further comprises single-stranded dna (ssdna).

3. The composition of

paragraphs

1 or 2 wherein the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the guide rna (grna).

4. The composition of any of paragraphs 1-3 wherein said dsDNA is activated dsDNA.

5. The composition of any of paragraphs 1-4, wherein said dsDNA is a dsDNA probe and has a luminescent/chromogenic group and a quencher group thereof.

6. The composition of paragraph 2 wherein the ssDNA is a ssDNA probe and has a luminescent/chromogenic group and a quencher group thereof.

7. The composition of paragraphs 5 or 6 wherein both ends of said dsDNA probe or said ssDNA probe are conjugated with a luminescent/chromogenic group and a quencher thereof.

8. The composition of any of paragraphs 1-7, wherein the small molecule has a molecular weight of 50-5000 daltons, preferably 50-1000 daltons.

9. The composition of any of paragraphs 1-8, wherein the small molecule is a heavy metal ion, toxin, drug, metabolite, contaminant, or a breakdown product thereof.

10. The composition of any of paragraphs 1-9, wherein the presence of said small molecule triggers the cleavage activity of CRISPR/Cas12a by said aTF and said dsDNA.

11. The composition of any of paragraphs 1 to 10, wherein the transcription factor binding site has a length of 10-40bp, preferably 15-25bp, more preferably 17-19 bp.

12. The composition of any of paragraphs 1-11, wherein said recognition agent comprises an allosteric transcription factor that is immobilized on a medium and/or dsDNA that is immobilized on a medium.

13. The composition of any of paragraphs 1-12, wherein the recognition agent comprises an allosteric transcription factor that can be immobilized on a medium.

14. The composition of any of paragraphs 1-13, wherein the medium is selected from the group consisting of: nitrocellulose or nylon membranes, affinity column chromatography matrices, magnetic beads, solid fillers, microcrystalline cellulose, or commercially available protein immobilization media.

15. The composition of paragraph 13 wherein the recognition agent comprises an allosteric transcription factor that is expressed fused to a microcrystalline cellulose domain that is immobilized on microcrystalline cellulose.

16. The composition of any of paragraphs 1-15, wherein the dsDNA is 20-80bp, more preferably 55-65bp, most preferably 58-60bp in length.

17. The composition of any of paragraphs 1-16, wherein the recognition reagent and the transduction reagent are provided in separate forms and react in different spaces.

18. The composition of any of paragraphs 6-17, wherein said ssDNA probe is any single-stranded DNA of 10-30bp in length

19. The composition of any of paragraphs 6-17, wherein said ssDNA probe is any single-stranded DNA of 20bp in length.

20. The composition of any of paragraphs 1-12, wherein the luminescent/chromophore is selected from the group consisting of: luminescent organic compounds, luminescent inorganic compounds, fluorophores, nanoparticles, quantum dots, and chromophores, and combinations thereof.

21. The composition of paragraph 1, wherein the composition comprises an identification agent and a transduction agent, wherein:

the recognition reagent comprises an allosteric transcription factor (aTF) and an activating double stranded DNA (activating dsDNA) comprising the binding site of aTF, the recognition site PAM of the CRISPR/Cas12a system and a sequence at least partially complementary to the guide RNA, and

22. the composition of paragraph 1, wherein the composition comprises an identification agent and a transduction agent, wherein:

the recognition reagent comprises aTF and a double-stranded DNA probe (dsDNA probe), the dsDNA probe comprises the binding site of aTF, a recognition site PAM of a CRISPR/Cas12a system and a sequence at least partially complementary to a guide RNA (gRNA), and both ends of the dsDNA probe are respectively conjugated with a luminescent/chromogenic group and a quencher thereof, and

23. The composition of paragraph 1, wherein the composition is in the form of a biosensor.

24. The composition of paragraph 23, wherein the biosensor comprises an identification element and a transducing element, wherein,

the recognition element comprises an allosteric transcription factor (aTF) and a double-stranded dna (dsDNA) comprising the binding site of aTF, the recognition site PAM of the CRISPR/Cas12a system, and a sequence at least partially complementary to the guide RNA; and the transducing element comprises the CRISPR/Cas12a system,

wherein, the biosensor also contains a luminescent/chromogenic group.

25. The composition of paragraph 24 wherein the transducing element further comprises single stranded dna (ssdna).

26. The composition of paragraphs 24 or 25 wherein the CRISPR/Cas12a system comprises a CRISPR/Cas12a protein and the guide rna (grna).

27. The composition of any of paragraphs 24-26, wherein said dsDNA is activated dsDNA.

28. The composition of any of paragraphs 24-26 wherein said dsDNA is a dsDNA probe and has a luminescent/chromogenic group and a quencher group thereof.

29. The composition of paragraph 25 wherein the ssDNA is a ssDNA probe and has a luminescent/chromophore and a quencher thereof.

30. The composition of paragraphs 28 or 29 wherein both ends of said dsDNA probe or said ssDNA probe are conjugated with a luminescent/chromogenic group and a quencher thereof.

31. The composition of paragraph 24 wherein the recognition element comprises an allosteric transcription factor (aTF) and an activating double stranded DNA (activating dsDNA) comprising the binding site of aTF, the recognition site of the CRISPR/Cas12a system (i.e., protospacer adjacent motif, PAM) and a sequence at least partially complementary to guide RNA, and

32. the composition of paragraph 24 wherein the recognition element comprises an allosteric transcription factor (aTF) and a double-stranded DNA probe (dsDNA probe) comprising a binding site for the allosteric transcription factor, a recognition site PAM for the CRISPR/Cas12a system, and a sequence that is at least partially complementary to a guide rna (grna), and both ends of the dsDNA probe are conjugated with a light/chromophore and its quencher, respectively, and

33. A composition as in any of paragraphs 1-32, wherein the recognition reagent and the transduction reagent are provided in separate forms and react in different spaces.

34. The composition of paragraph 21 wherein the allosteric transcription factor and dsDNA, the CRISPR/Cas12a protein, guide rna (grna), and ssDNA probes are provided as a single reagent; or

The allosteric transcription factors and activating dsDNA, the CRISPR/Cas12a protein, guide rna (grna), and ssDNA probes are provided as separate reagents.

35. The composition of paragraph 22 wherein the allosteric transcription factor and dsDNA probe, the CRISPR/Cas12a protein, and a guide rna (grna) are provided as a single agent; or

The allosteric transcription factor and dsDNA probes, the CRISPR/Cas12a protein and guide rna (grna) are provided as separate reagents.

36. A kit for detecting a small molecule comprising the composition of any of paragraphs 1-35.

37. The kit of paragraph 36, wherein the kit further comprises a detection device for detecting an optical signal.

38. The kit of paragraph 38 or 39, wherein the kit further comprises a delivery means or device, a wash buffer, a dilution buffer, a stop buffer, a data carrier, a standard, or a container describing instructions for use, and combinations thereof, for use in operating the biosensor of any of paragraphs 1-35.

39. A method of detecting a small molecule in a test sample, the method comprising detecting a small molecule in the test sample using the composition of any one of paragraphs 1-35 or the kit of any one of paragraphs 36-38.

40. The method of paragraph 39 wherein the method includes:

(1) mixing and incubating the sample to be detected and the recognition reagent; (2) isolating free dsDNA fragments from the resulting mixture; (3) adding the separated free dsDNA to a transduction reagent and detecting the resulting optical signal; and, (4) detecting the presence or amount of the small molecule based on the generated signal.

41. The method of paragraph 39 or 40, wherein the method comprises:

(1) mixing and incubating the sample to be tested, aTF and dsDNA; (2) isolating free dsDNA fragments from the resulting mixture; (3) adding the isolated free dsDNA to the CRISPR/Cas12a protein and guide RNA and detecting the resulting optical signal; and (4) detecting the presence or amount of the small molecule based on the generated signal.

42. The method of paragraph 39 or 40, wherein the method comprises:

(1) mixing and incubating the test sample, aTF and the activated dsDNA fragments;

(2) isolating free activated dsDNA fragments from the mixture in step (1);

43. The method of paragraph 39 or 40, wherein the method comprises:

(1) mixing and incubating the sample to be tested, aTF and dsDNA probe;

(2) isolating free dsDNA probes from the mixture in step (1);

44. The method of paragraphs 42 or 43 wherein in step (1) the molar ratio of said allosteric transcription factor to said activating dsDNA is ≥ 5: 1; the molar ratio of the allosteric transcription factor to the dsDNA probe is more than or equal to 5: 1.

45. The method of paragraphs 42 or 43 wherein in step (1) said allosteric transcription factor is first mixed with said activated dsDNA to form a complex, and then unbound activated DNA is removed and said test sample is added; or, the allosteric transcription factor is mixed with the dsDNA probe to form a complex, then the unbound dsDNA probe is removed, and then the sample to be detected is added.

46. The method of any of paragraphs 40-45, wherein in step (1), said mixing is at room temperature for more than 1 min.

47. The method of any of paragraphs 40-46, wherein in step (1) said allosteric transcription factor is an allosteric transcription factor that can be immobilized on a medium.

48. The method of paragraph 47 wherein prior to step (1), a mediator is added to immobilize the allosteric transcription factor that is immobilized on a mediator onto the mediator.

49. The method of any of paragraphs 39-48, wherein said sample to be tested is from an environment, a subject, a food product, drinking water, a cosmetic product or a feed.

50. The method of paragraph 49 wherein the 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 isolates, pleural fluid, cystic fluid, tissue, saliva.

51. The method of paragraphs 40-50, wherein said sample to be tested is subjected to a pretreatment of enrichment, extraction and/or purification prior to step (1).

52. The method of any of paragraphs 40-51, wherein in step (2) the separation is performed by filtration, centrifugation, sedimentation, magnetic bead method, chromatography, affinity column method.

53. The method of any of paragraphs 40-52, wherein in step (3) the detection is by fluorescence or colorimetric analysis.

54. The method of any of paragraphs 40-53, wherein in step (4) the optical signal is analyzed based on a reference level, said reference level being the level of a sample that does not contain said small molecule or the level of a sample that contains a known amount of a small molecule.

55. Use of a composition of any of paragraphs 1-35 in the preparation of a kit for detecting a small molecule.

56. The use of paragraph 55 wherein the kit is used for environmental pollution monitoring, food and cosmetic quality control and disease diagnosis.

References in Table 1

1S.P.Wilkinson,A.Grove,J.Biol.Chem.,2004,279,51442-51450.

2K.Ike,Y.Arasawa,S.Koizumi,S.Mihashi,S.Kawai-Noma,K.Saito,D.Umeno,ACSSynth.Biol.,2015,4,1352-1360.

3Y.Nakada,Y.Jiang,T.Nishijyo,Y.Itoh,C.D.Lu,J.Bacteriol.,2001,183,6517-6524.

4F.A.Cerda-Maira,G.Kovacikova,et.al.,2013,195,307-317.

5D.Lechardeur,B.Cesselin,et.al.,J.Biol.Chem.,2012,287,4752-4758.

6T.Shimada,A.Ishihama,S.J.Busby,D.C.Grainger,Nucleic Acids Res.,2008,36,3950-3955.

7H.B.Guo,A.Johs,et.al.,J.Mol.Biol.,2010,398,555-568.

8C.Xu,W.Shi,B.P.Rosen,J.Biol.Chem.,1996,271,2427-2432.

9K.P.Yoon,T.K.Misra,S.Silver,J.Bacteriol.,1991,173,7643-7649.

10H.J.Kim,J.W.Lim,et.al.,Biosens.Bioelectron.,2016,79,701-708.

11W.Wang,T.Yang,et.al.,ACS Synth.Biol.,2016,5,765-773.

12P.Vargas,A.Felipe,et.al.,Mol.Plant Microbe Interact.,2011,24,1207-1219.

13N.Noguchi,K.Takada,et.al.,J.Bacteriol.,2000,182,5052-5058.

14M.Folcher,R.P.Morris,et.al.,J.Biol.Chem.,2001,276,1479-1485.

15S.Ghosh,C.M.Cremers,et.al.,Mol.Microbiol.,2011,79,1547-1556.

16A.Roy,A.Ranjan,Biochemistry,2016,55,1120-1134.

17Y.Kim,G.Joachimiak,et.al.,J.Biol.Chem.,2016,291,13243-13256.

18T.Li,K.Zhao,Y.Huang,et.al.,Appl.Environ.Microb.,2012,78,6009-6016.

19A.Hernández,F.M.Ruiz,A.Romero,J.L.Martínez,PLoS Pathog.,2011,7,e1002103.

20L.Cuthbertson&J.R.Nodwell,Microbiol.Mol.Biol.Rev.,2013 77,440-475.

21S.Kitani et al.Proc.Natl.Acad.Sci.USA2011,108,16410-16415.

Examples

The dsDNA fragments (nucleotide sequences shown in SEQ ID NOs: 5-7), guide RNAs (nucleotide sequences shown in SEQ ID NOs: 9-11), primers (shown in Table 2), and probes (nucleotide sequence shown in SEQ ID NO:8, labeled with FAM at the 5 'end and BHQ at the 3' end) used in the following examples were synthesized by Kinsley.

The media and reagents used in the following examples are as follows:

LB culture medium: 1% of NaCl, 1% of peptone and 0.5% of 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 with a miscellaneous buffer solution: 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 equilibration 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

TABLE 2 primers used in examples 1 to 2

Example 1 uric acid biosensor

In this example, the allosteric transcription factor HucR and the activating dsDNA fragment (dsDNA) were used_hucR(ii) a Wherein a HucR binding site (in which a sequence complementary to the gRNA is contained) and a CRISPR/Cas12a recognition site (PAM)) are included as recognition elements, and CRISPR/Cas12a, gRNA, and single-stranded DNA (ssDNA; two ends of the probe are marked with FAM fluorophore and BHQ fluorescence quenching group as probes) as a transduction element, and a biosensor platform capable of sensing uric acid is constructed.

The HucR protein is derived from Deinococcus radiodurans (Deinococcus radiodurans) repressor protein, and the amino acid sequence of the HucR protein can be shown as SEQ ID NO. 1. As shown in FIG. 1, HucR binds dsDNA in the absence of Uric Acid (UA)_hucRFormation of the HucR action site on HucR-dsDNA_hucRThe composite. Uric acid binds specifically to HucR in the presence of uric acid, allowing dsDNA_hucRObtaining dsDNA in free form from HucR Release_hucRFree form dsDNA_hucRThe CRISPR/Cas12a system is activated, causing DNA probe cleavage, generating a fluorescent signal. Thus, uric acid concentration in the sample can be determined by monitoring the fluorescent signal.

Cloning, expression and purification of HucR-CBD fusion protein

Using pET35b plasmid (Wuhan vast Ling Biotech Co., Ltd.) containing CBD gene (the coded amino acid sequence is shown as SEQ ID NO: 4) as template, and obtaining CBD fragment by amplification of primer CBD-F/HucR-CBD-R; single cleavage with Xho I (reaction system: 10 Xcutmarst buffer 2. mu.L; plasmid 1. mu.g; Xho I1. mu.L; H)₂O is supplemented to 20 mu L; reaction at 37 ℃ for 1h) on the pET23b-HucR plasmid (pre-constructed: the pET23b plasmid and the HucR gene fragment were subjected to double digestion with Nde I and Xho I (reaction system: 10 Xcutmarst buffer 2. mu.L; plasmid/HucR gene fragment 1. mu.g; Xho I1. mu.L; Nde I1. mu.L; H)₂O is supplemented to 20 mu L; reaction at 37 ℃ for 1h), purification and recovery, and ligation by T4DNA ligase (reaction system: 10x T4ligasebuffer 1 μ L; 50-100ng of plasmid; 50-100ng of the HucR gene fragment; h₂O is supplemented to 10 mu L; reacting for 1h) at 22 ℃ and carrying out linearization treatment; the CBD fragment and linearized pET23b-hucR were assembled with Exmax using the seamless cloning (single-fragment ligation) kit (Tolo Biotechnology, Shanghai) according to the instructions provided by the manufacturer to obtain pET23b-HucR-CBD plasmid, which was confirmed as positive by PCR and sequencing. 10ng of plasmid pET23b-HucR-CBD was transformed into Escherichia coli BL21 (Tiangen Biochemical technology Co., Ltd., Beijing) by calcium transfer, and Amp resistance was selected as a positive clone. Single colonies were picked and inoculated on 100mL LB (Amp)⁺) The medium was shaken at 200rpm at 37 ℃ overnight (. about.12 h). 2.5mL (1 v/v% inoculum size) of the culture was transferred to 250mL of LB (Amp)⁺) The culture medium was added with IPTG at a final concentration of 0.2mM, and the mixture was subjected to low-temperature induction at 16 ℃ and 180rpm overnight, followed by centrifugation at 4 ℃ and 9,000rpm for 15min, and the cells were collected and washed with a washing buffer. Each 2L of the mycelia was suspended in 50mL of pre-cooled binding buffer. Placing the suspension on ice, ultrasonically cracking thallus (ultrasonic cell crusher, ultrasonic for 3s, intermittent for 5s, 30W, 20min), centrifuging at 4 deg.C and 12000rpm for 30min, and collecting supernatant and repeating the above centrifuging operation to clarify. The lysate was passed over a HisTrap FF column (GE Healthcare) and eluted with elution buffer. The peak fractions were collected and dialyzed (dialysis membrane from Shanghai works; dialysis buffer). The dialyzed solution was then applied to a HiTrap QHP column (GE Healthcare) and eluted with elution buffer. The peak fractions were collected and concentrated using a ultrafiltration tube (millicore). The concentrate was applied to a HiLoad 16/600Superdex 200pg column for flash protein liquid chromatography (FPLC; AKTAExporer 100, GE Healthcare). The purified HucR-CBD protein was verified by SDS-PAGE and was determined to be 3mg/mL using the Bradford method.

Activity analysis of HucR-CBD fusion protein

To analyze the effect of the CBD domain on the activity of the HucR protein, gel retardation assay (EMSA) was used to compare the molecular weights of the CBD domain and HucR to dsDNA_HucRIs analyzed. Use of primer dsDNA_HucR-1 and dsDNA_HucRAnnealing (pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, 75 cycles, 1 ℃ reduction per cycle) to obtain 60 bp-sized dsDNA_(HucR)For EMSA analysis. The experimental conditions and data acquisition methods for EMSA were determined according to Wang et al, molecular microbiology, 2011, 82 (1): 236-250. 0, 25, 50, 100, 200nM of HucR-CBD was combined with 40nM dsDNA_HucRMixing (in 20mL buffer solution: 10mM Tris-HCl (pH7.5), 100mM KCl, 1mM EDTA, 0.1mM DTT, 5% v/v glycerol, 0.01mg/mL bovine serum albumin, the binding of dsDNA to aTF and the action of effector on dsDNA and aTF are all performed in the buffer solution), keeping away from light at room temperature for 15min, performing electrophoresis on 1.5% agarose gel, and detecting the fusion protein HucR-CBD and dsDNA_HucRIn combination with (1).

The results are shown in FIG. 2B, when HucR-CBD dsDNA_HucR(ii) dsDNA at 200nM:40nM (i.e., 5:1)_HucRCan be completely combined by HucR-CBD, and the fusion expression with the CBD structural domain can not influence the function of the HucR.

Recognition and response of HucR-CBD fusion protein to uric acid

For analysis of effector uric acid on HucR-CBD-dsDNA_HucRDissociation of the Complex into HucR-CBD dsDNA_HucRDifferent concentrations of uric acid (0, 0.05, 0.5, 5, 50, 500 μ M) were added to the 5:1 incubation system and dsDNA was detected by EMSA_HucRDissociation of (3). The results are shown in FIG. 2C, with increasing uric acid concentration, free dsDNA_HucRAre increasing continuously.

Preparation of Cas12a protein and gRNAs

pET28TEV-Cas12a plasmid (LbCas12a, Tou Luo harbor) was constructed, and pET28TEV-Cas12a plasmid was transformed into E.coli BL21 competent cells in the same procedure as the procedure for expression and purification of HucR-CBD fusion protein, and after induction of protein expression by 0.2mM IPTG, the cells were collected by centrifugation and disrupted by ultrasonication using an ultrasonicator, and cell lysate was collected, purified and concentrated to obtain Cas12a purified protein with a concentration of 1 mg/mL.

Annealing of the T7 primer to the crRNA-HucR primer in Taq DNA polymerase buffer (20. mu.L of body)Comprises the following steps: 2 μ L of 10 × TaqDNA polymerase buffer; 9 μ L of T7 primer; crRNA-HucR primer 9. mu.L; PCR procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, 1 ℃ per cycle, 70 cycles) to synthesize a template for crRNA (i.e., gRNA). Then transcribed into crRNA using HiScribe T7 Rapid high Performance RNA Synthesis kit (NEB) and RNA Clean&Concentrator^TM-5(ZymoResearch) for purification. The concentration of the resulting crRNA was measured using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific) at 2000-3000 ng/. mu.L.

5. Detection of uric acid

2mg of microcrystalline cellulose (Shanghai Biotech; cat. No. 9004-34-6) was washed twice with Tris-HCl buffer, and mixed with the fusion protein HucR-CBD (200nM) in NEB

Buffer, mixed and incubated for 10 minutes at room temperature. Centrifugation at 7000rpm at room temperature and discarding the supernatant, NEB was used

Unbound protein was removed by 3 buffer washes and immobilized protein was quantified using the Bradford assay kit (tiangen biochem corporation); add 100nM dsDNA_HucRReaction at room temperature for-9 min, centrifuging at room temperature at 7000rpm to discard the supernatant, washing more than or equal to 1 time to remove unbound dsDNA_HucR(ii) a To each of the obtained dsDNA-protein-cellulose complexes, 500. mu.M of uric acid and its analogs (adenine, guanine, hypoxanthine) were added, reacted at room temperature for 15min, and centrifuged at 7000rpm to take the supernatant. The supernatant (which may contain dsDNA in free form) is added_HucRdsDNA in free form at 40nM_HucRAs a positive control) a mixture of Cas12a, gRNA and FAM/BHQ-modified ssDNA (SEQ ID NO: 9) was added (50nM Cas12a, 50nM gRNA and 250nM ssDNA in 20. mu.L NEB

In buffer), mixed and immediately placed in BMG Clariostar microplate reader (BMG Labtech, UK) for reaction at 37 deg.C, measured under 480nm excitation light and 520nm emission light andthe fluorescence intensity of the reaction system was recorded. The results are shown in FIG. 3C, positive control (i.e., dsDNA in free form)_HucR) The group with uric acid obtained the strongest fluorescence intensity, while the group with uric acid analogs (adenine, guanine, hypoxanthine) had a fluorescence intensity of less than 5% of that of the positive control group. And the results are similar to those of EMSA (fig. 3B), demonstrating that the uric acid biosensor of the present invention can accurately recognize and detect small molecules.

6. Analysis of biosensor Performance

6.1 Linear detection Range of dsDNA

To investigate the effect of different concentrations of dsDNA on the change in fluorescence intensity, different concentrations of dsDNA were taken_HucR(1, 5, 10, 25, 50, 100pM) A mixture of Cas12a, gRNA and FAM/HQ-modified ssDNA (SEQ ID NO: 9) was added (50nM Cas12a, 50nM gRNA and 250nM ssDNA in 20. mu.L NEB

In buffer), mixed and immediately placed in a BMG CLARIOstar enzyme-labeling instrument (BMG Labtech, UK) for reaction at 37 ℃, and the fluorescence intensity of the reaction system is measured and recorded under 480nm excitation light and 520nm emission light. As shown in FIG. 4A (left column), the fluorescence intensity of the reaction system increased with increasing concentration of dsDNA. In order to analyze the linear relationship between the dsDNA and the change of the fluorescence intensity of the reaction system, the slope of the interval of linear increase of the fluorescence value is taken, the dsDNA concentration is taken as the abscissa, and the corresponding slope is taken as the ordinate to plot 4A (right column), so that the linear detection range of the dsDNA is 1-25 pM.

6.2 Linear detection Range of uric acid

In order to investigate the influence of uric acid with different concentrations on the change of fluorescence intensity, uric acid with different concentrations was detected by the above method, and the fluorescence intensity of the reaction system was measured and recorded, and the result is shown in fig. 4B (left column), wherein as the concentration of uric acid increases, the content of free dsDNA increases, and the fluorescence intensity also increases. The change of the fluorescence intensity of the reaction system gradually increases with the increase of uric acid, and the linearity between the uric acid concentration and the change of the fluorescence intensity of the reaction system is analyzedIn the relationship, the slope of the fluorescence value linear increasing interval is taken, the uric acid concentration is taken as the abscissa, the corresponding slope is taken as the ordinate, 4B (right column) is plotted, the linear detection range of the uric acid is 25-500nM, and the regression equation is as follows: y 10.7x +1289.8, R²0.992. The detection limit of the sensor of the embodiment on uric acid is in nM level, while the existing uric acid biosensor is usually in μ M level (such as the uric acid biosensor described in patent 201810224843.8, 1.71 μ M), and it can be seen that the biosensor of the present invention has excellent detection sensitivity (as low as 25 nM).

6.3 analysis of clinical blood samples

Equal amounts of serum and chloroform were mixed and shaken vigorously, centrifuged at 10,000g for 10 minutes at room temperature, and the supernatant was collected. Uric acid in a serum sample was detected using the uric acid biosensor of this example using the method described above. To avoid errors in aspiration, the serum sample supernatant was diluted 10-fold with water and assayed using 1. mu.L of the dilution. Since HPLC was able to detect uric acid content in serum very accurately, samples were subjected to HPLC as a control. HPLC conditions: AgilentSB-Aq column (4.6 mm. times.150 mm, 5 μm, 1 mL/min); water was used as eluent and was detected at 284nm (UV detector; Agilent1260 definition II LC System). The uric acid concentrations measured by HPLC were plotted on the ordinate and the uric acid concentrations measured by the biosensor detection method of this example were plotted on the ordinate (fig. 5A), and it can be seen that the results measured by the two methods are linearly related (y is 0.978x, R is 0.978 ×)²0.993), which indicates that the detection method of the present invention has high reliability.

Further, comparing the detection method of the present embodiment with a background uric acid detection kit used clinically, the uric acid concentration measured by background is also plotted as ordinate and the uric acid concentration measured by the biosensor detection method of the present embodiment is plotted as ordinate (fig. 5B), and it can be seen that the results measured by the two methods are linearly related (y is 1.023x, R is R, and R is R, y is 1.023x, R is R²0.983), also indicating that the detection method of the present invention has a high degree of confidence.

The normal range of uric acid in human serum is 166.4-546.7 μ M, but since the biosensor of the present invention is very sensitive, only a very small amount of blood sample (about 1 μ L) is required to obtain accurate results (FIG. 5C), so that the reaction system has a small volume, and the detection can be performed on 96-well plates or even 384-well plates. In addition, the method can obtain results only in 15-25 minutes and at most in 25 minutes, not only saves time, but also has low cost and simple and convenient operation, and is very suitable for high-throughput detection of uric acid in samples such as blood and the like.

6.4 comparison with other detection methods

To further evaluate the advantages of the uric acid biosensor of the present example, it was compared with various detection methods reported in the prior art, and the results are summarized in table 3.

TABLE 3 comparison of different Small molecule detection methods

RT-qPCR: real-time fluorescent quantitative PCR; RCA: rolling circle amplification; RPA: recombinase polymerase amplification; PGM: a blood glucose meter; ISDA: isothermal strand displacement amplification; the clinical method comprises the following steps: a Backman kit; Cat-SMelor: the biosensor of the present invention; y: applicable; n: is not applicable; NA: is not obtained.

Moreover, for the uric acid biosensor implemented in the embodiment, since the fluorescence signal is relatively stable, the fluorescence half-life of the biosensor exceeds 200min, which provides researchers or other users with more time to record and analyze the experimental results.

In conclusion, the uric acid biosensor has excellent detection sensitivity, higher reliability, time and cost saving and simple operation, is very suitable for the in vitro detection of small molecules requiring high flux, high speed and high sensitivity, and has wide application prospect in laboratories, medical treatment and industrial application.

Example 2 Tetracycline biosensor and p-hydroxybenzoic acid (p-HBA) sensor

To verify the universality of the sensor and method of the invention, the concept of example 1 was adopted, designing the transmission in response to tetracycline and p-HBA, respectivelyA sensor is provided. Biosensors responsive to tetracycline utilize the allosteric transcription factor, TetR, and double-stranded activating DNA fragments (dsDNA)_TetR(ii) a Wherein a TetR binding site and a CRISPR/Cas12a recognition site (PAM)) are included as recognition elements, and CRISPR/Cas12a, gRNA and single-stranded DNA (ssDNA; FAM fluorophore and BHQ fluorescence quencher labeled at both ends as a probe) as a transducer element. Biosensors responding to p-HBA use the allosteric transcription factor HosA and a double stranded activating DNA fragment (dsDNA)_HosA(ii) a Wherein the HosA binding site and CRISPR/Cas12a recognition site (PAM)) are contained as recognition elements, and the CRISPR/Cas12a, gRNA and single-stranded DNA (ssDNA; FAM fluorophore and BHQ fluorescence quencher labeled at both ends as a probe) as a transducer element.

TetR-CBD fusion proteins and HosA-CBD fusion proteins were prepared according to the method and procedure of example 1, tetracycline biosensors and p-HBA sensors were constructed, and the performance of the p-HBA sensors was analyzed.

1. Analysis of p-HBA sensor Performance

1.1 assay of HosA-CBD fusion protein Activity

Different concentrations (0, 25, 50, 100, 200nM) of HosA-CBD were mixed with 40nM dsDNA_HosA(the nucleotide sequence is shown as SEQ ID NO: 6), reacting at room temperature for 15min, and performing EMSA to detect HosA-CBD and dsDNA_HosAIn combination with (1). The results are shown in FIG. 2D when the HosA-CBD dsDNA is present_HosAWhen the ratio is 5:1, dsDNAHosA can be completely bound by HosA-CBD, and fusion expression with CBD domain does not affect the functional activity of HosA.

1.2 recognition and response of the fusion protein HosA-CBD to p-HBA

For analysis of the effectors p-HBA on HosA-CBD and dsDNA_HosADissociation of the complexes into HosA-CBD dsDNA_HosAdsDNA detection by EMSA with different concentrations (0, 0.18, 1.8, 18, 180, 1800. mu.M) of p-HBA added to the reaction system at 5:1_HosADissociation of (3). The results are shown in FIG. 2E, with increasing p-HBA concentration, free dsDNA_HosAAre increasing continuously.

1.3 detection of p-HBA

Taking 2mg microcrystalline cellulose (Shanghai Biotech; product number 9004-34-6) and buffering with Tris-HClThe washes were washed twice and mixed with the fusion protein HosA-CBD (200nM) in NEB

Unbound protein was removed by 3 buffer washes and immobilized protein was quantified using the Bradford assay kit (tiangen biochem corporation); add 100nM dsDNA_HosAReaction at room temperature for 10 minutes, centrifugation at 7000rpm at room temperature to discard the supernatant, and washing 3 times to remove unbound dsDNA_HosA(ii) a To each of the resulting dsDNA-protein-cellulose complexes, 1.8mM of p-HBA and its analogs (p-hydroxyphenylethanol, p-aminobenzoic acid (p-ABA), methylparaben (p-MHB), p-hydroxybenzyl alcohol (p-HBnOH) were added, reacted at room temperature for 15min, and the supernatant was centrifuged at 7000rpm_HosAdsDNA in 1.8mM free form_HosAAs a positive control) a mixture of Cas12a, gRNAs (nucleotide sequence shown in SEQ ID NO: 10) and FAM/BHQ-passed ssDNA (sequence shown in SEQ ID NO: 9) (50nM Cas12a, 50nM gRNAs and 250nM ssDNA in 20. mu.L NEB)

In buffer), mixed and immediately placed in a BMG CLARIOstar enzyme-labeling instrument (BMG Labtech, UK) for reaction at 37 ℃, and the fluorescence intensity of the reaction system is measured and recorded under 480nm excitation light and 520nm emission light. The results are shown in FIG. 3E, positive control (i.e., dsDNA in free form)_HosA) And the p-HBA group obtained the strongest fluorescence intensity, while the p-HBA analogue (p-hydroxyphenyl ethanol, p-ABA, p-MHB, p-HBnOH) group had a fluorescence intensity of less than 5% of that of the positive control group. And the results are similar to those of EMSA (fig. 3D), demonstrating that the p-HBA biosensor of the present invention is able to accurately recognize and detect small molecules.

1.4 Linear detection Range of p-HBA

To investigate the effect of different concentrations of p-HBA on the change in fluorescence intensity, different concentrations (1.8, 9, 18, 90, 180, 900, 1800nM) of p-HBA were measured and the fluorescence intensity of the reaction system was determined and recorded, as shown in FIG. 4C (left column), free dsDNA with increasing p-HBA concentration_HosAThe content is increased, and the change of fluorescence intensity is improved. In order to analyze the linear relationship between the p-HBA concentration and the change of the fluorescence intensity of the reaction system, the slope of the linearly increasing interval of the fluorescence value is taken, the p-HBA concentration is taken as the abscissa, the corresponding slope is taken as the ordinate to plot 4C (right column), and the linear detection range of the dsDNA is 9-180nM, and the regression equations are respectively as follows: y 6.3x +656.6, R²0.992. Therefore, the PHBA sensor performance according to the present invention also has an excellent detection limit (down to 1.8 nM).

Therefore, the sensor and the method have universality, can detect specific small molecular compounds at high sensitivity, high speed and high flux by means of various allosteric transcription factors, are simple to operate and low in cost, and have wide application prospects in laboratories and industrial applications.

Sequence listing

<110> university of eastern China college of sciences microbial research institute

<120> biosensor based on CRISPR/Cas12a system, kit and application thereof in small molecule detection

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Arg Ala Ile Ala Asp Lys Pro Gly Ile Glu Gln Val Ala Leu Ile Glu

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Arg Arg Phe Val Trp Leu Thr Ala Glu Gly Glu Lys Val Leu Ala Ala

100 105 110

Ala Ile Pro Ile Gly Asp Ser Val Asp Ala Glu Phe Leu Gly Arg Leu

115 120 125

Ser Gly Glu Glu Gln Glu Leu Phe Met Gln Leu Val Arg Lys Met Met

130 135 140

Ser Lys

145

<210>3

<211>207

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu

1 5 10 15

Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln

20 25 30

Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys

35 40 45

Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His

50 55 60

Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg

65 70 75 80

Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly

85 90 95

Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr

100 105 110

Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu

115 120 125

Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys

130 135 140

Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr

145 150 155 160

Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu

165 170 175

Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu

180 185 190

Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser

195 200 205

<210>4

<211>157

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Met Ser Val Glu Phe Tyr Asn Ser Asn Lys Ser Ala Gln Thr Asn Ser

1 5 10 15

Ile Thr Pro Ile Ile Lys Ile Thr Asn Thr Ser Asp Ser Asp Leu Asn

20 25 30

Leu Asn Asp Val Lys Val Arg Tyr Tyr Tyr Thr Ser Asp Gly Thr Gln

35 40 45

Gly Gln Thr Phe Trp Cys Asp His Ala Gly Ala Leu Leu Gly Asn Ser

50 55 60

Tyr Val Asp Asn Thr Ser Lys Val Thr Ala Asn Phe Val Lys Glu Thr

65 70 75 80

Ala Ser Pro Thr Ser Thr Tyr Asp Thr Tyr Val Glu Phe Gly Phe Ala

85 90 95

Ser Gly AlaAla Thr Leu Lys Lys Gly Gln Phe Ile Thr Ile Gln Gly

100 105 110

Arg Ile Thr Lys Ser Asp Trp Ser Asn Tyr Thr Gln Thr Asn Asp Tyr

115 120 125

Ser Phe Asp Ala Ser Ser Ser Thr Pro Val Val Asn Pro Lys Val Thr

130 135 140

Gly Tyr Ile Gly Gly Ala Lys Val Leu Gly Thr Ala Pro

145 150 155

<210>5

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tactgagcca tgtatccagg tcattgtact tagatgtcta cctaagctct gacagttcca 60

<210>6

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

tactgagcca tgtatccagg tcattgcgtt cgtatacgaa cagtagctct gacagttcca 60

<210>7

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

tactgagcca tgtatccagg tcattgcgtt cgtatacgaa cagtagctct gacagttcca 60

<210>8

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gattagcgta cgcacgttac 20

<210>9

<211>38

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gaauuucuac uguuguagau uacuuagaug ucuaccua 38

<210>10

<211>38

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gaauuucuac uguuguagau cguucguaua cgaacagu 38

<210>11

<211>38

<212>RNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gaauuucuac uguuguagau ucccuaucag ugauagag 38

<210>12

<211>20

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 510 15

Gly Gly Gly Gly

20

<210>13

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gtggtggtgg tggtgctcga gtggtgctgt accaagaact t 41

<210>14

<211>85

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

tgtgaaagtg ggtctctcga gtctggcggc ggcggctctg gcggcggcgg ctctggcggc 60

ggcggcatgt cagttgaatt ttaca 85

<210>15

<211>86

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ctcgaacagg gtgttctcga gtctggcggc ggcggctctg gcggcggcgg ctctggcggc 60

ggcggcatgt cagttgaatt ttacaa 86

<210>16

<211>85

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

tgtgaaagtg ggtctctcga gtctggcggc ggcggctctg gcggcggcgg ctctggcggc 60

ggcggcatgt cagttgaatt ttaca85

<210>17

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

gaaattaata cgactcacta taggg 25

<210>18

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

tggaactgtc agagcttagg tagacatcta agtacaatga cctggataca tggctcagta 60

<210>19

<211>60

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

tggaactgtc agagctactg ttcgtatacg aacgcaatga cctggataca tggctcagta 60

<210>20

<211>59

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

tggaactgtc agagcttctc tatcactgat agggacaaat gacctggata catggctca 59

<210>21

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

gttgtaaaac gacggccagt 20

<210>22

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

acacaggaaa cagctatgac 20

<210>23

<211>62

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

taggtagaca tctaagtaat ctacaacagt agaaattccc tatagtgagt cgtattaatt 60

tc 62

<210>24

<211>62

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

actgttcgta tacgaacgat ctacaacagt agaaattccc tatagtgagt cgtattaatt 60

tc 62

<210>25

<211>59

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

ctctatcact gatagggaat ctacaacagt agaaattccc tatagtgagt cgtattaat 59

Claims

wherein the composition further comprises a luminescent/chromophore group.

2. The composition of claim 1, wherein the composition is in the form of a biosensor.

3. A kit for detecting a small molecule comprising the composition of claim 1 or 2.

4. A method for detecting a small molecule in a test sample, the method comprising detecting a small molecule in the test sample using the composition of claim 1 or 2 or the kit of claim 3.

5. Use of a composition according to claim 1 or 2 in the manufacture of a kit for the detection of small molecules.

6. The use according to claim 5, wherein the kit is used for environmental pollution monitoring, food and cosmetic quality control and disease diagnosis.