CN117126923A - Molecular detection method - Google Patents

Abstract

The invention relates to a molecular detection method that uses multi-channel single molecule mechanics technology to simultaneously manipulate multiple molecular probes to detect analytes; both ends of the molecular probes are connected to a carrier through labeled coupling functional groups; the carrier is used to detect molecules The probe exerts pulling force. The detection process includes binding force and detection force: under the binding force, the molecular probe binds to the analyte; under the detection force, the difference in length or length change curve of the molecular probe with or without the analyte is used as the signal intensity to detect whether There is the analyte, the analyte concentration, the binding speed and binding strength of the analyte to the specific structure. The invention is suitable for detecting the following analytes, such as proteins, drugs, small molecules, DNA, RNA and their complexes, etc.

Description

A molecular detection method

Technical field

The invention belongs to the field of biology and new medical technology, and in particular relates to a molecular detection method.

Background technique

Nucleic acid probes are a type of molecular probe composed of nucleic acid molecules or nucleic acid-like molecules. They can specifically sense the analyte in the component to be measured through a specific molecular recognition mechanism, and transfer the molecules to the analyte through a specific signal transduction method. Recognition events are converted into fluorescent signals or other electrochemical signals and reported.

Single-molecule mechanical manipulation technology is a technology used to study the structure and interactions of biomolecules such as proteins and nucleic acids, and can be used for molecular detection. Compared with traditional methods, technologies based on single molecule manipulation, such as optical tweezers, acoustic tweezers, magnetic tweezers, liquid flow manipulation, etc., can use the applied pulling force or the extension length of the probe molecules as identification signals, so there is no need to use Signal transduction methods such as fluorescence quantification not only avoid the need for a signal output domain during probe design, but also greatly reduce the complexity of probe design. On the other hand, measurement of a single molecule can theoretically detect a single analyte, which is the ultimate solution to solve the sensitivity deficiency of classic nucleic acid probes.

However, due to the limitation of molecular diffusion speed, the binding speed of the analyte to the single-molecule probe is limited, which results in a very long detection time for the analyte with a very low concentration, which greatly reduces the detection sensitivity. In addition, how to improve detection resolution, distinguish multiple analytes that bind to nucleic acid probes, detect multiple analytes simultaneously, detect the binding speed, intensity and concentration of analytes, and correctly detect background signals in biological or clinical samples. Detecting the analyte, avoiding false positives, and detecting mutations in the nucleic acid of the analyte, especially single-base mutations, are all technical problems that need to be solved.

Contents of the invention

In order to solve the above technical problems, the present invention provides a molecular detection method that uses multi-channel single molecule mechanics technology to simultaneously manipulate multiple molecular probes to detect analytes, and can simultaneously detect multiple analytes in complex samples.

The molecular detection method provided by the invention uses multi-channel single molecule mechanics technology to simultaneously manipulate multiple molecular probes to detect the analyte; both ends of the molecular probe are connected to a carrier through labeled coupling functional groups; the carrier is used to detect the molecular probe. The needle exerts pulling force.

On the basis of the above technical solution, the molecular probe is a tandem nucleic acid probe, and the monomer of the tandem nucleic acid probe contains a target sequence; under a certain range of pulling force, the target sequence can form a specific structure that binds the analyte; tandem Nucleic acid probes are prepared by rolling circle amplification or rolling circle transcription. This method can detect the presence or absence of the analyte, the concentration of the analyte, the binding speed and binding strength of the analyte to the specific structure by detecting the length difference of the molecular probe or the difference in length change curve of the analyte as signal intensity. Tandem nucleic acid probes increase detection sensitivity by increasing the number of specific structures.

Based on the above technical solution, rolling circle amplification includes the following steps:

The initiation of rolling circle amplification: rolling circle amplification primers, DNA polymerase and rolling circle amplification template are required; the rolling circle amplification template contains the starting sequence and the complementary sequence of the target sequence; the starting sequence can be combined with the rolling circle amplification primer;

Extension of rolling circle amplification: All necessary nucleotides are used as substrates and amplification is continued for a period of time based on the desired degree of polymerization of the tandem nucleic acid probes.

Based on the above technical solution, rolling circle transcription includes the following steps:

Initiation of rolling circle transcription: RNA polymerase and rolling circle transcription template are required; the rolling circle transcription template contains the promoter sequence and the complementary sequence of the target sequence; the promoter sequence is a sequence that binds RNA polymerase; in this step, the newly generated RNA is transcribed This marker has coupling functional groups;

Elongation of rolling circle transcription: Transcription takes all necessary nucleotides as substrates and continues for a period of time depending on the desired degree of polymerization of the tandem nucleic acid probe.

Based on the above technical solution, the extension step is performed by fixing on the carrier.

Based on the above technical solutions, rolling circle amplification or rolling circle transcription involves a process of pausing and then restarting.

On the basis of the above technical solution, the rolling circle amplification or rolling circle transcription reaction reagent lacks at least one of the necessary nucleotide substrates, and the polymerase pauses when the missing nucleotide substrate needs to be used.

On the basis of the above technical solutions, the detection process using multi-channel single molecule mechanics technology includes binding force and detection force: under the binding force, the target sequence forms a specific structure and can bind to the analyte; under the detection force, the analyte is detected test object;

When the detection force is a constant force, record the length of the tandem nucleic acid probe when there is no analyte as the reference length. After changing from the binding force to the detection force, calculate the difference between the length of the tandem nucleic acid probe and the reference length as the signal intensity.

When the detection force is a gradient force, record the length change curve of the tandem nucleic acid probe when there is no analyte as the reference curve. During the sample detection process, the difference between the length change curve of the tandem nucleic acid probe and the reference curve is calculated as the signal intensity under the detection force;

According to the signal intensity, the presence of the analyte in the sample, the concentration of the analyte, the binding strength and binding speed of the analyte to the specific structure are detected.

On the basis of the above technical solution, the degree of polymerization of the multiple tandem nucleic acid probes is uniform; the uniformity standard is that the mean square error of the degree of polymerization is less than 20% of the average degree of polymerization.

Based on the above technical solution, the molecular detection includes: simultaneously manipulating a group of multiple tandem nucleic acid probes, and the target sequences of various tandem nucleic acid probes in the group are sequences that identify sequence polymorphisms.

Specific structures are defined by their ability to bind to the test substance, including but not limited to single-stranded DNA or single-stranded RNA, hairpin structure, double-stranded structure, nano-origami, aptamer, G4, i-motif, nucleic acid Three-chain structure, etc. The specific structure may be an unfolded structure formed by the target sequence under a larger pulling force, or it may be a folded structure formed by the target sequence under a smaller pulling force.

The present invention combines tandem nucleic acid probes with multi-channel single molecule mechanics technology, simultaneously manipulates multiple tandem nucleic acid probes, and uses the length difference or length change curve difference of the tandem nucleic acid probes under different binding conditions with the test substance to obtain the following detection Results: (i) Whether there is a analyte; (ii) The concentration of the analyte; (iii) The binding strength of the analyte to the nucleic acid probe; (iv) The binding speed of the analyte to the nucleic acid probe; (v) ) Improve the detection resolution of analytes; (vi) Distinguish multiple analytes bound to nucleic acid probes; (vii) Detect multiple analytes simultaneously; (viii) Correctly detect background signals in biological or clinical samples Detect the analyte; (ix) use multiple probes as controls to avoid false positives; (x) detect mutations in the nucleic acid analyte, especially single base mutations.

The method for synthesizing multiple tandem nucleic acid probes of the present invention is as follows:

Tandem nucleic acid probes can be synthesized by rolling circle amplification or rolling circle transcription. Both ends of the probe are coupled to the carrier respectively. The function of the carrier is to exert pulling force on the probe.

Preferably, the carrier can be a flow cell or microspheres.

Simultaneously manipulate multiple probes using multichannel single-molecule mechanics techniques.

Preferably, the multi-channel single molecule mechanics technology is magnetic tweezers.

The degree of polymerization of multiple tandem nucleic acid probes measured in parallel is uniform.

Preferably, the criterion for uniformity is that the mean square error of the degree of polymerization is less than 20% of the average degree of polymerization.

The degree of polymerization of the tandem nucleic acid probe is greater than 500.

The uniformity of polymerization of tandem nucleic acid probes can be improved by starting, pausing and restarting the process. Among them, pausing and then restarting after starting can use the method of assembling rolling circle amplification or rolling circle transcription complexes at low temperature. At this time, the activity of the polymerase is extremely low, so that the reaction system can be started simultaneously after returning to the reaction temperature. Polymerase inhibitors can also be used to inhibit amplification or transcription activity after rolling circle amplification or rolling circle transcription is initiated, so that it can be started simultaneously after the inhibitor is removed. In the embodiments of the present invention, the method of lacking at least one necessary nucleotide in the reaction substrate of the circle amplification or rolling circle transcription initiation step is specifically adopted to realize the process of pausing and restarting.

The monomer of the tandem nucleic acid probe includes a sequence forming a hairpin structure, and the target sequence is located in the stem region of the hairpin structure. Under binding force, the hairpin structure opens into a single strand and exposes the target sequence. The target sequence in the single-stranded state can bind to the test substance.

The specific preparation method of tandem nucleic acid probes is as follows:

1. Synthesis of tandem nucleic acid probes through rolling circle amplification

When synthesizing tandem nucleic acid probes through rolling circle amplification, the rolling circle amplification process can be performed in situ on a carrier or in a solution. Compared with the method of synthesis in solution, the advantages of in-situ synthesis are: first, it is convenient to replace reaction reagents; second, since there is no mutual interference between rolling circle amplification complexes during the synthesis process, on the one hand, new synthesis can be prevented The entanglement between probe molecules affects the structural integrity of the probe molecules. On the other hand, it helps to synthesize probes with a higher degree of polymerization.

The technical details of rolling circle amplification in situ synthesis of tandem nucleic acid probes are:

Technical details 1: Through a coupling reaction, the rolling circle amplification primer is coupled to the carrier through the coupling functional group 1.

Preferably, the carrier is a flow cell or microspheres.

The coupling between rolling circle amplification primers and flow cells or microspheres can be covalent binding or affinity connection.

Preferably, the coupling functional group 1 modified by the rolling circle amplification primer is a sulfhydryl group, and the modification is located at the 5' end of the primer sequence and can be connected to an SMCC-modified flow cell or microsphere.

Preferably, the rolling circle amplification primers are attached to the SMCC modified flow cell surface.

Rolling circle amplification primers can be designed based on the probe sequence and can be combined with the promoter sequence in the rolling circle amplification template.

Preferably, the rolling circle amplification primers used in the examples are all SH_hp.

Technical detail two: Make a closed-structure circular rolling circle amplification template through enzyme ligation reaction.

The closed structure circular amplification template contains the complementary sequence of the promoter sequence and the target sequence. The initiating sequence is a sequence that can be combined with a rolling circle amplification primer; the target sequence can form a specific structure that binds the test substance.

Preferably, the target sequence is located in the stem region of the probe hairpin structure.

The closed-structure circular rolling circle amplification template can be composed of two hairpin-structured single-stranded nucleic acids with sticky ends connected by ligase; it can also be connected by a single-stranded nucleic acid in the presence of bridge DNA. Enzymes are linked end to end.

Preferably, the rolling circle amplification templates in the embodiments are composed of two hairpin-structured single-stranded DNAs with sticky ends connected by ligase.

Preferably, the ligase is commercial T4 DNA ligase.

Preferably, the length of the sticky end is 6 bases.

Preferably, the 5' end of the sticky end is phosphorylated. The phosphorylation modification at the 5' end can be added during artificial synthesis or using polynucleotide phosphorylase.

Preferably, the phosphorylation modifications at the 5' end are added during artificial synthesis.

The sequence of the rolling circle amplification template can be designed based on the analyte.

Preferably, the two hairpin structures used in the embodiment are T1 and T2 respectively. The hairpin structure T1 contains the initiating sequence, and the hairpin structure T2 contains the complementary sequence of the target sequence.

Technical detail three: Synthesize tandem nucleic acid probes through rolling circle amplification.

Preferably, the DNA polymerase used in the rolling circle amplification process is an isothermal polymerase.

Preferably, the warm polymerase is commercial phi29 DNA polymerase.

Preferably, the reaction temperature during rolling circle amplification is 30°C.

The nucleotide substrate used in the extension step of rolling circle amplification can be a modified nucleotide substrate, such as methylation-modified dATP or dCTP, or it can be an unmodified nucleotide substrate.

Preferably, the nucleotide substrate used in the rolling circle amplification extension step is dNTP without modification.

In the present invention, the tandem nucleic acid probe synthesized by rolling circle amplification contains a coupling functional group 2 at its 3' end, and the labeling of the coupling functional group 2 can be achieved by DNA polymerase or terminal transferase.

Preferably, the labeling of the coupling functional group 2 at the 3' end is achieved by incorporating a certain proportion of a dNTP modified with the coupling functional group 2 after rolling circle amplification and extension.

Preferably, the dNTP modified with coupling functional group 2 is biotin-labeled dUTP.

Preferably, the incorporation ratio of the biotin-labeled dUTP is that the ratio of biotin-labeled dUTP to unlabeled dTTP is equal to 1:9.

The 3’ end of the probe is connected to the carrier through the coupling functional group 2.

Preferably, the carrier is a flow cell or microspheres. Therefore, both ends of the probe are connected to the flow cell or microspheres, respectively.

Technical detail four: The rolling circle amplification process can be synchronized to make the degree of polymerization of multiple synthesized tandem nucleic acid probes relatively uniform.

Rolling circle amplification also includes the process of starting rolling circle amplification, pausing and then restarting the amplification.

In the present invention, the rolling circle amplification process is synchronized by adding a defective substrate to the reaction reagent in the initial step of rolling circle amplification. The defective substrate lacks at least one of the nucleotide substrates necessary for rolling circle amplification. Therefore, the DNA polymerase pauses when the missing nucleotide substrate needs to be used.

Preferably, the defective substrate lacks a dNTP nucleotide substrate.

Preferably, the one dNTP nucleotide substrate is dTTP.

When synthesizing tandem nucleic acid probes through rolling circle amplification, the rolling circle amplification process can also be performed in solution. Compared with the rolling circle amplification process performed in situ on the surface of the carrier, the technical details are different:

One of the differences in technical details is that rolling circle amplification extension is not performed on a vector.

The second difference in technical details is that when synchronizing the rolling circle amplification process, the rolling circle amplification complex needs to be recovered after the DNA polymerase pauses amplification.

2. Synthesis of tandem nucleic acid probes through rolling circle transcription

When synthesizing tandem nucleic acid probes through rolling circle transcription, the rolling circle transcription process can be performed in situ on the carrier or in solution. Compared with the method of synthesis in solution, the advantages of in-situ synthesis are: first, it is convenient to replace reaction reagents; second, since there is no mutual interference between rolling circle transcription complexes during the synthesis process, on the one hand, it can prevent new synthetic probes from The entanglement between needle molecules affects the structural integrity of the probe molecules. On the other hand, it helps to synthesize probes with a higher degree of polymerization.

The technical details of rolling circle transcription in situ synthesis of tandem nucleic acid probes are:

Technical details one: synthesize a closed-structure circular rolling circle transcription template through enzyme ligation reaction and annealing reaction. The closed structure circular rolling circle transcription template contains the promoter sequence and the complementary sequence of the target sequence. Among them, the initiation sequence has a double-stranded structure and can bind to RNA polymerase.

The enzyme ligation reaction can be formed by connecting two single-stranded nucleic acids with a hairpin structure with sticky ends through a ligase, or it can be formed by connecting a single-stranded nucleic acid end-to-end through a ligase in the presence of bridging DNA. .

Preferably, in the embodiment, the enzymatic ligation reaction of the rolling circle transcription template is formed by ligating a single-stranded nucleic acid end-to-end through a ligase in the presence of bridging DNA.

Preferably, the bridging DNA is Bridge1, and the ligase is commercial T4 DNA ligase.

The annealing reaction is to anneal the complementary sequence of the initiation sequence on the enzyme-linked circular single-stranded template to form a circular rolling circle transcription template with a local double-stranded structure.

Preferably, the complementary sequence of the promoter sequence is T7_C.

The sequence of the closed structure circular rolling circle transcription template can be designed according to the type of RNA polymerase and the analyte.

Preferably, in the embodiment, the starting sequence of the rolling circle transcription template is a T7 promoter sequence, and the RNA polymerase is T7 RNA polymerase.

The target sequence of the closed-structure circular rolling circle transcription template is a sequence that can be folded into a hairpin structure, or a sequence that can be folded into a G-quadruplex structure, an aptamer structure, a nano-origami structure, a drug target-specific structure, etc.

The single-stranded nucleic acid is T3.

Preferably, the T3 is T3_G4. The 5' end of T3_G4 is phosphorylated. The phosphorylation modification of the 5' end can be added during artificial synthesis or modified using polynucleotide phosphorylase.

Preferably, the phosphorylation modification of the 5' end is added during artificial synthesis.

Technical detail two: through in-situ rolling circle transcription on the vector, multiple tandem nucleic acid probes with a certain degree of polymerization are synthesized. The tandem nucleic acid probes have a coupling functional group 3 at the 5' end and a coupling functional group at the 3' end. Functional group 4.

In the present invention, for the tandem nucleic acid probe synthesized by rolling circle transcription, the 5' end coupling functional group 3 is labeled by introducing a substrate modified by the coupling functional group 3 in the initial step of rolling circle transcription.

Preferably, the coupling functional group 3 is digoxigenin, and the substrate modified by the coupling functional group 3 is digoxigenin-labeled UTP, which is introduced into the nascent transcript at the initial step of rolling circle transcription, so that the tandem The 5' end of the nucleic acid probe is labeled with digoxigenin.

After the initiation of rolling circle transcription, the complex formed by the digoxigenin-labeled nascent RNA transcript and the rolling circle transcription complex is purified. The purified complex is coupled to the carrier through a coupling reaction.

Preferably, the carrier is a flow cell, and the digoxigenin antibody is modified on the flow cell.

Various nucleotide substrates necessary for rolling circle transcription are added to allow the rolling circle transcription reaction to continue, and transcription continues for a period of time based on the required degree of polymerization of the tandem nucleic acid probe. The various nucleotide substrates necessary for the elongation step of rolling circle transcription can be modified nucleotides, such as methylated ATP or methylated CTP, or they can be unmodified nuclei. glycosides.

Preferably, the various nucleotide substrates necessary for the elongation step of rolling circle transcription are nucleotides without modifications.

In the present invention, the modification of the 3' end coupling functional group 4 of the tandem nucleic acid probe synthesized by rolling circle transcription can be achieved by RNA polymerase or terminal transferase.

Preferably, the 3' end coupling functional group 4 is achieved by incorporating a certain proportion of a nucleotide substrate modified with the coupling functional group 4 after rolling circle transcription elongation.

Preferably, the nucleotide substrate modified with coupling functional group 4 is biotin-labeled UTP.

Preferably, the incorporation ratio of the biotin-labeled UTP is that the ratio of biotin-labeled UTP to unlabeled UTP is equal to 1:9.

Technical detail three: By synchronizing the rolling circle transcription process, the degree of polymerization of multiple synthesized tandem nuclear probes is relatively uniform.

Preferably, the relative homogeneity of the degree of polymerization means that the mean square error of the degree of polymerization is less than 20% of the average degree of polymerization.

In the present invention, the rolling circle transcription process is synchronized by adding a defective substrate to the reaction reagent in the initial step of rolling circle transcription. The defective substrate lacks at least one of the nucleotide substrates necessary for rolling circle transcription. Therefore, the RNA polymerase pauses when the missing nucleotide substrate needs to be used.

Preferably, the defective substrate lacks an NTP nucleotide substrate.

Preferably, the missing NTP nucleotide substrate is GTP.

When tandem nucleic acid probes are synthesized by rolling circle transcription, all steps of the rolling circle transcription process can also be performed in solution. Compared with the rolling circle transcription process performed in situ on the vector, the technical details differ in that before the elongation step of rolling circle transcription, the complex formed by the nascent RNA transcript and the rolling circle transcription complex is purified. No prior coupling to a support is required.

The second aspect is single molecule mechanics detection.

The present invention uses multiple tandem nucleic acid probes prepared and combines multi-channel single molecule mechanics technology to implement molecular detection. The multi-channel single molecule mechanics technology that can be used can be single molecule magnetic tweezers technology, multi-channel optical tweezers technology, single molecule acoustic tweezers technology, fluid force technology, etc.

Preferably, the multi-channel single molecule mechanics technology used is single molecule magnetic tweezers technology. The technical details are:

Technical details 1: Through a coupling reaction, the two ends of the tandem nucleic acid probe are coupled to the flow cell and microspheres respectively.

Preferably, the coupling reaction refers to coupling the coupling functional group 2 at the 3' end of the tandem nucleic acid probe synthesized through rolling circle amplification or the coupling functional group 4 at the 3' end of the tandem nucleic acid probe synthesized through rolling circle transcription. On flow cells or microspheres.

Preferably, the 3' end of the tandem nucleic acid probe is connected to the magnetic microsphere.

Preferably, the magnetic microspheres are commercial superparamagnetic spheres M-270 (Dynabeads) with a diameter of 2.8 microns, and the surface modification of the magnetic spheres is streptavidin modification. The beneficial effect of using this kind of superparamagnetic ball M-270 is that this kind of magnetic ball has a uniform saturation magnetic moment, so when it is manipulated at the same time, it can exert a uniform pulling force on the connected probes.

Technical detail two: By annealing the single strand complementary to the spacer sequence, the non-functional spacer sequence in the tandem nucleic acid probe is changed from a single strand to a double strand. The non-functional spacer sequence is a sequence that does not participate in the detection of the analyte. The beneficial effect is to increase the overall hardness of the probe molecules and reduce Brownian motion noise during the detection process.

Preferably, for tandem nucleic acid probes synthesized through rolling circle amplification, the single strand with complementary spacer sequences is Flank1; for tandem nucleic acid probes synthesized through rolling circle transcription, the single strand with complementary spacer sequences is Flank2.

Technical details three: Set the mechanical stretching mode during the detection implementation process. Mechanical stretching mode contains detection force and bonding force.

The detection force can be constant force. Under the detection force, record the length of the probe without the object to be measured as the reference length. During the sample detection process, after changing from binding force to detection force, the difference between the length of the probe and the reference length is calculated as the signal intensity. According to the signal intensity, the presence of the analyte in the sample, the concentration of the analyte, the binding speed and binding strength of the analyte to the specific structure are detected.

Preferably, when the signal intensity is greater than 3 times the mean square error of the background noise, it is determined that the object to be measured is present.

On the basis of the above technical solution, the concentration of the analyte is calculated based on the signal intensity and the standard curve during detection by pre-measurement of the standard curve of the concentration of the analyte and the signal intensity.

On the basis of the above technical solution, using different detection powers, different signal strengths can be obtained. When the detection force is less than the binding force, the smaller the detection force when the signal intensity is reduced to greater than 3 times the mean square error of the background noise, the stronger the binding; when the detection force is greater than the binding force, when the signal intensity is reduced to greater than the background The greater the detection power when 3 times the noise mean square error, the stronger the binding.

The detection force can be a gradient force. Under the detection force, record the length change curve of the probe when there is no object to be measured as the reference curve. During the sample detection process, the difference between the length change curve of the probe and the reference curve is calculated as the signal intensity under the detection force. According to the signal intensity, the presence of the analyte in the sample, the concentration of the analyte, the binding speed and binding strength of the analyte to the specific structure are detected.

Preferably, when the signal intensity is greater than 3 times the mean square error of the background noise, it is determined that the object to be measured is present.

On the basis of the above technical solution, the concentration of the analyte is calculated based on the signal intensity and the standard curve during detection by pre-measurement of the standard curve of the concentration of the analyte and the signal intensity.

On the basis of the above technical solution, when the detection force is less than the binding force, the detection force gradually becomes smaller. When the signal intensity decreases to greater than 3 times the mean square error of the background noise, the smaller the detection force, the stronger the binding; when the detection force When it is greater than the binding force, the detection force gradually becomes larger. When the signal intensity decreases to greater than 3 times the mean square error of the background noise, the greater the detection force is, indicating the stronger the binding.

On the basis of the above technical solution, the time to maintain the binding force, that is, the binding time, can be changed. According to the relationship between signal intensity and binding time, under the condition of known concentration of the analyte, the binding speed of the analyte to the specific structure is measured.

On the basis of the above technical solution, when multiple probes are used, and the probes contain different specific structures, the binding strength and binding speed of the test substance to different specific structures can be detected simultaneously.

After a round of detection is completed, a probe regeneration step can be added. In this step, the nucleic acid probe can be restored to its baseline state without binding to the analyte for the next round of detection.

Preferably, the pulling force exerted on the probe during the regeneration step is the regeneration force.

The force application steps in the detection process in the embodiment are as follows: detection force, recording the length or length curve of the probe when there is no object to be measured as the reference length or reference length curve; binding force, the target sequence on the probe forms a specific structure, Identify and bind the analyte. The longer it stays under the binding force, the more the analyte is bound to the probe and the stronger the binding signal. Detection force records the length or length of the probe after the analyte is bound. curve and calculate the difference between the length or length curve and the reference length or reference length curve as the signal intensity; with renewed force, the analyte bound to the probe is completely dissociated, and the probe returns to the original unbound analyte. state.

3. Measure multiple tandem nucleic acid probes simultaneously

Multiple tandem nucleic acid probes can have the same sequence and be used to detect the same analyte. The beneficial effect is that one measurement is equivalent to multiple measurements, saving detection time and reagents, and reducing false positives and false negatives. Parallel errors between multiple inspections are avoided. The beneficial effect is also to increase the number of specific structures. For example, when the degree of polymerization is 1000 and 100 probes are detected simultaneously, it is equivalent to increasing the number of specific structures by 100,000 times.

Multiple tandem nucleic acid probes can differ in sequence. Therefore, multiple analytes can be detected simultaneously.

Preferably, the plurality of tandem nucleic acid probes can simultaneously detect nucleic acids of multiple pathogens that cause fever.

Multiple tandem nucleic acid probes can differ in sequence. Therefore, the binding strength and binding speed of the same analyte to different specific structures can be detected.

Multiple tandem nucleic acid probes can also differ in sequence. Therefore, a probe that does not contain the target sequence can be used as a negative control during the detection process. The beneficial effect is that when detecting biological or clinical samples, negative control probes are used to remove background noise caused by irrelevant molecules in the sample.

Groups of multiple nucleic acid probes can be manipulated simultaneously. The target sequences of various nucleic acid probes in the group are sequences for identifying sequence polymorphisms, and the length of the target sequences is 12-26 bases. Its beneficial effect is that it can be used for base mutation detection of nucleic acid test substances. The mutation may be a single base mutation or a multiple base mutation, a base conversion or base transversion mutation, or a base deletion or base insertion mutation.

Preferably, the number of base mutations in the test substance is 1-2. The position of the mutated base on the test substance corresponds to the middle part of the probe target sequence.

Description of the drawings

Figure 1 is a schematic diagram showing the use of two hairpin-structured single-stranded DNA with sticky ends to construct a circular rolling circle amplification template through a ligation reaction. The template contains complementary sequences of the priming sequence and the target sequence.

Figure 2 is a schematic diagram showing the synthesis of tandem nucleic acid probes with uniform polymerization degree by in-situ synchronized rolling circle amplification on the flow cell surface. ① Couple the rolling circle amplification primer containing coupling functional group 1 to the surface of the flow cell; ② Anneal the rolling circle amplification template to the primer; ③ Add DNA polymerase and defective substrate to assemble the rolling circle amplification complex. Start rolling circle amplification and then pause; ④ Add the four nucleotide substrates dNTP mix necessary for rolling circle amplification, restart rolling circle amplification and continue amplification for a period of time according to the required degree of polymerization of the tandem nucleic acid probe; ⑤ Add the nucleotide substrate biotin-dUTP labeled with the coupling functional group 2, and continue amplification so that the 3' end of the tandem nucleic acid probe is labeled with biotin; ⑥ Single-stranded Flank1 that is complementary to the spacer sequence through annealing, Change the non-functional spacer sequence of the tandem nucleic acid probe from single-stranded to double-stranded; ⑦ Connect the 3' end of the tandem nucleic acid probe to the magnetic microsphere M-270 through biotin labeling.

Figure 3. Under a constant pulling force of 12 pN, the polymerization degree and uniformity of the tandem nucleic acid probes are detected. The polymerization degree is 1470±154.

Figure 4 By repeatedly using tandem nucleic acid probes specific to the analyte N_20, the real-time length change curves of the tandem nucleic acid probes under binding force were measured in the measurement solution, the analyte N_20 at a concentration of 100 pM, and Flank1 at a concentration of 10 nM.

Figure 5 takes a perfectly matched nucleic acid analyte and a nucleic acid analyte with a single base mutation as an example to verify that the gradually decreasing gradient force can be used as the detection force to measure the binding strength of the analyte to the tandem nucleic acid probe. . The test substance N-16 has stronger binding strength than N-16-G9C.

Figure 6 uses the standard curve method to determine the concentration of the analyte miR-122 in liver cancer and adjacent tissues. Figure A shows the relationship between the binding signal intensity and concentration of the analyte miR-122 and the specific probe; Figure B shows the quantitative results of the analyte miR-122 in the sample.

Figure 7 is a schematic diagram showing the use of known analyte standards to sequentially determine the species of each probe in a tandem nucleic acid probe set with a uniform degree of polymerization.

Figure 8 is a schematic diagram showing the principle of using multiple tandem nucleic acid probes with uniform polymerization degrees to detect base mutations in the analyte.

Figure 9 Multiple tandem nucleic acid probes with uniform polymerization degree detect and distinguish let-7 family members in serum whose sequences differ only by 1-2 bases: let-7a, let-7b, let-7c and let-7d.

Figure 10 takes let-7b and let-7c as examples to illustrate that the use of tandem nucleic acid probes and multi-channel single molecule mechanics technology can detect rare mutations with mutation probability as low as 1 in 10,000.

Figure 11 Multiple tandem nucleic acid probes with uniform polymerization degrees detect novel coronavirus nucleic acid fragments in saliva and identify virus subtypes.

Figure 12 is a schematic diagram showing the process of using bridge DNA and ligation reactions to connect single-stranded linear DNA templates end-to-end and construct a circular rolling circle transcription template.

Figure 13 is a schematic diagram showing the synthesis of multiple tandem nucleic acid probes with uniform polymerization degrees by in-situ synchronized rolling circle transcription on the surface of the flow cell. ① Prepare the rolling circle transcription complex labeled with the coupling functional group 3 and can be extended synchronously; ② Connect the rolling circle transcription complex to the surface of the flow cell through the coupling functional group 3; ③ Add the four nucleosides necessary for rolling circle transcription amplification Acid substrate NTPmix, restart rolling circle transcription and continue transcription for a period of time according to the required degree of polymerization of the tandem nucleic acid probe; ④ Add biotin-UTP, a nucleotide substrate labeled with coupling functional group 4, and continue transcription to make the tandem nucleic acid probe The 3' end of the nucleic acid probe is labeled with biotin; ⑤ By annealing the single-stranded Flank2 complementary to the spacer sequence, the non-functional spacer sequence in the tandem nucleic acid probe is changed from single-stranded to double-stranded; ⑥ By biotin labeling, the single-stranded Flank2 is complementary to the spacer sequence. The 3' end of the tandem nucleic acid probe is connected to the magnetic microsphere M-270.

Figure 14 is a schematic diagram showing the use of multiple tandem nucleic acid probes with uniform polymerization degrees to measure the impact of small molecule analytes on the stability of the G-quadruplex structure.

Figure 15 Uniform polymerization tandem nucleic acid probe detects that the small molecule ligand PDS can significantly increase the stability of the RNAG quadruplex structure formed by human telomeric repeat sequences.

Detailed ways

The present invention utilizes a method that can simultaneously synthesize multiple tandem nucleic acid probes to obtain multiple tandem nucleic acid probes with uniform polymerization degrees, and uses these tandem nucleic acid probes combined with multi-channel single molecule mechanics technology to design a nucleic acid detection method. Device, using this nucleic acid detection device, multiple molecules can be detected simultaneously.

The features and advantages of the present invention will be further understood through the detailed description of the following embodiments in conjunction with the accompanying drawings. The provided embodiments are only partial illustrations of the method of the present invention and do not limit the remaining contents disclosed in the present invention in any way. In the examples where the specific experimental conditions are not clearly stated in the experimental protocols, all operations should be carried out in accordance with the operating steps in the corresponding product instructions. The reagents, consumables and instruments used in the examples can be purchased from commercial companies unless otherwise specified.

Example 1: Using two hairpin-structured single-stranded DNAs with sticky ends, a circular rolling circle amplification template was constructed through a ligation reaction.

Specific steps are as follows:

Step 1: Design and synthesize card issuance structure templates T1 and T2_1, whose structures are shown in Figure 1.

The sequence of template T1 is SEQ ID NO:1:

5’-CTGGACGTCGATCGTTCGTGAAGTCAACATCGAACTCTCTTGCCAATCCTGGTCGTGATTCCATTCTATCGATGTTGACTTCACGAACGATCGAC-3’;

The loop portion after folding into a hairpin structure contains the initiation sequence.

The sequence of template T2_1 is SEQ ID NO:2:

5’-GTCCAGATGCATCTCAAGGTCTAGTGCTGCTTTTGCAGCACTAGACCTTGAGATGCAT-3’;

The double-stranded part after folding into a hairpin structure contains the complementary sequence of the target sequence.

Step 2: Folding of template T1 and T2_1 hairpin structures. Templates T1 and T2_1 with a concentration of 2 μM were incubated at 95°C for 10 minutes and then quickly cooled on ice.

Step three: Enzyme ligation reaction to synthesize closed-structured single-stranded circular DNA. The templates T1 and T2_1 that form the hairpin structure are mixed in equal amounts, reacted with T4 DNA ligase at 4°C for 12 hours for enzyme ligation reaction, and then reacted at 65°C for 10 minutes to inactivate the T4 DNA ligase.

Step 4: Purify and recover closed-structured single-stranded circular DNA. The ligation product in step three is recovered through an ultrafiltration tube and treated with DNA exonuclease III and DNA exonuclease I in sequence to digest all linear or nick-containing by-products to obtain a higher purity single unit with a closed structure. Stranded circular DNA is used as rolling circle amplification template T_1.

Step 5: Polyacrylamide gel electrophoresis detects the products of steps 2, 3 and 4 respectively. The gel concentration was 8%, and the gel was electrophoresed at 100V for 40 minutes, then soaked and stained with the nucleic acid dye Gel-Red, and imaged by the Bio-Rad gel imaging system.

Example 2: In-situ synchronized rolling circle amplification on the flow cell surface was used to synthesize tandem nucleic acid probes with uniform polymerization degrees, as shown in Figure 2. Specific steps are as follows:

Step 1: Couple the rolling circle amplification primer to the surface of the flow cell through sulfhydryl labeling. The preparation of the flow cell and the amination process of the flow cell surface were carried out according to the classic experimental procedures for single-molecule experiments. Then the amine-thiol cross-linker Sulfo-SMCC (Thermo Scientific ^TM ) was added and reacted under standard conditions for 30 minutes. Remove the free amine-sulfhydryl cross-linking agent Sulfo-SMCC, add rolling circle amplification primer SH_hp with a concentration of 0.1pM dissolved in PBS buffer, and react at room temperature for 1 hour. The sequence of rolling circle amplification primer SH_hp is SEQ ID NO:3:

5’-GCATTAGGAAGCAGCCCAGTAGTAGGATCACGACCAGGATTG-3’. Add BSA passivation solution to seal the active functional group of the rolling circle amplification primer that has not been successfully coupled to the surface of the flow cell.

Step 2: In-situ synchronized rolling circle amplification on the surface of the flow cell synthesizes multiple tandem nucleic acid probes with biotin labeling at the 3' end and uniform polymerization. 1) Dilute the rolling circle amplification template T_1 with 3×PBS buffer to a concentration of 1ng/μL, add it to the flow cell coupled with the rolling circle amplification primer, and incubate at 37°C for 30 minutes to allow the rolling circle amplification template to The priming sequence fully anneals to the rolling circle amplification primer and forms a primer template linker. 2) Rinse away the free rolling circle amplification template, add reaction buffer containing defective substrates (dATP, dCTP and dGTP) and phi29 DNA polymerase, and incubate at 30°C for 5 minutes to allow phi29 DNA polymerase to fully recognize the primer template adapter. , forming a rolling circle amplification complex, initiating rolling circle amplification and pausing at the first encounter with base A on the template. 3) Rinse away the free phi29 DNA polymerase, add the reaction buffer containing the complete reaction substrate dNTP mix, restart the rolling circle amplification process, and continue incubating at 30°C for 15 minutes. 4) Add the reaction substrate mixed with biotin-labeled dUTP and continue incubating at 30°C for 1 minute to add biotin label to the 3' end of the tandem nucleic acid probe. The ratio of biotin-labeled dUTP to dTTP is equal to 1:9. 5) Add 30mM EDTA to terminate the rolling circle amplification reaction. 6) Add the spacer complementary sequence Flank1 at a concentration of 100 nM, whose sequence is SEQ ID NO: 4:

5’-CTCTTCTTGCCAATCCTGGTCGTGATTCCATTCT-3’. The spacer sequence connecting adjacent hairpin structures in a tandem nucleic acid probe with a uniform degree of polymerization is changed from a single strand to a double strand.

Step 3: Connect newly synthesized tandem nucleic acid probes with uniform polymerization degree to magnetic microspheres. The magnetic microsphere M-270 is diluted 100 times with 3×PBS buffer and then added to the flow cell. The surface of the magnetic microsphere M-270 is modified with streptavidin, which can be used with the biotin labeled at the 3' end of the tandem nucleic acid probe. coupling.

Step 4: Under a constant pulling force of 12pN, determine the molecular length of the tandem nucleic acid probe by measuring the distance between the magnetic microspheres coupled with the tandem nucleic acid probe and the surface of the flow cell. Based on the elastic parameters of the probe molecules in the measurement solution and measurement conditions, the degree of polymerization of the tandem nucleic acid probe was further calculated to be 1470±154, as shown in Figure 3.

Example 3: Detection of artificial sequence N_20 by tandem nucleic acid probes with uniform polymerization degree. Specific steps are as follows:

Step 1: According to Example 1, the hairpin sequences T1 and T2_1 are used to synthesize a circular rolling circle amplification template T_1 that can target the artificial sequence N_20. The sequence of N_20 is SEQ ID NO: 5: 5'-ATGCATCTCAAGGTCTAGTG-3'.

Step 2: Synchronize in-situ rolling circle amplification according to the steps described in Example 2 to synthesize a tandem nucleic acid probe with a uniform degree of polymerization that can specifically recognize N_20.

Step 3: Connect the tandem nucleic acid probes with a uniform degree of polymerization to the magnetic microspheres and place them in the single-molecule magnetic tweezers device. Set the mechanical stretching mode of the detection process to: the detection force (F1=10pN) stays for 5 seconds to determine the binding. In front of the analyte, the molecular extension length of the probe under the detection force is recorded as the reference length; the binding force (F2=20pN) stays for 20 seconds, and the target sequence on the probe molecule is exposed under the binding force, and the target sequence is recognized and bound. Test object; the detection force (F1=10pN) is held for 5 seconds, and after binding to the test object, the change in the extension length of the probe molecule under the detection force compared to the reference length is recorded, recorded as LB, as the binding of the test object Signal intensity; the regeneration force (F3=1pN) stays for 10 seconds to dissociate all the analytes bound to the probe, and the probe returns to the initial state of unbound analytes.

Step 4: Repeat the mechanical stretching mode in Step 3, using the measurement solution as a negative control, the artificial sequence N_20 at a concentration of 100pM as the experimental group, and Flank1 at a concentration of 10nM as the negative control, and record the tandem nucleic acid probes with uniform polymerization degrees. The real-time length change curve of the needle under the binding force of each group of samples. The results showed that the probe molecules showed obvious binding signals only in the presence of the analyte, as shown in Figure 4.

Example 4: Using the gradually decreasing gradient force as the detection force, measure the binding strength between the test substance and the tandem nucleic acid probe. Specific steps are as follows:

Step 1: According to the steps shown in Example 1 and Example 2, use hairpin structure templates T1 and T2_1 and in-situ synchronized rolling circle amplification to synthesize tandem nucleic acid probes with a uniform degree of polymerization. This probe can specifically identify the perfectly paired analyte N_16 and the analyte N_16_G9C with a single base mutation. The sequence of N_16 is SEQ ID NO: 6: 5'-ATCTCAAGGTCTAGTG-3'; the sequence of N_16_G9C is SEQ ID NO: 7: 5'-ATCTCAAGCTCTAGTG-3'.

Step 2: Connect the tandem nucleic acid probes with a uniform degree of polymerization to magnetic microspheres and place them in the single-molecule magnetic tweezers device. Set the mechanical stretching mode of the detection process to: the detection force (F1) gradually decreases from 13pN to 3pN. Determine the molecular length change curve of the probe molecule under the detection force before binding to the analyte, and use it as the benchmark curve; the binding force (F2 = 20pN) stays for 20 seconds; the target sequence on the probe molecule is exposed under the binding force, and the target sequence is identified And bind the analyte; the detection force (F1) gradually decreases from 13pN to 3pN. After binding the analyte, record the difference between the molecular length change curve of the probe molecule under the detection force and the reference curve, as the binding value of the analyte. Signal intensity LB; the regeneration force (F3=1pN) stays for 10 seconds to dissociate all the analytes bound to the probe, and the probe returns to the initial state of unbound analytes.

Step 3: Repeat the mechanical stretching mode in Step 2, use the measurement solution as a negative control, and measure the binding strength of the perfectly matched test substance N_16 and the test substance N_16_G9C with a single base mutation to the tandem nucleic acid probe. The results showed that the mutation of a single base in the analyte significantly reduced the binding strength of the analyte to the target sequence of the tandem nucleic acid probe, as shown in Figure 5.

Example 5: Tandem nucleic acid probes with uniform polymerization degree were used to detect the expression changes of microRNA (miR-122) in liver cancer tissues and adjacent tissues. Specific steps are as follows:

Step 1: According to Example 1, hairpin sequences T1 and T2_2 are used to synthesize a circular rolling circle amplification template T_2 that can target miR-122.

The sequence of T2_2 is SEQ ID NO:8:

5’-GTCCAGAGTGTGACAATGGTGTTTGCTGCTTTTGCAGCAAACACCATTGTCACAC T-3’;

The sequence of miR-122 is SEQ ID NO:9: 5'-UGGAGUGUGACAAUGGUGUUUG-3' (RNA).

Step 2: Synchronize in situ and synthesize a tandem nucleic acid probe with a uniform degree of polymerization that can specifically recognize miR-122 according to the steps described in Example 2.

Step 3: Connect the tandem nucleic acid probes with a uniform degree of polymerization to the magnetic microspheres and place them in the single-molecule magnetic tweezers device. Set the mechanical stretching mode of the detection process to: the detection force (F1=9pN) stays for 5 seconds to determine the binding. Before miR-122, the molecular extension length of the probe under the detection force is recorded as the reference length; the binding force (F2=20pN) stays for 3 minutes, and the target sequence on the probe molecule is exposed under the binding force, and the target sequence is recognized and bound. Detect the analyte miR-122 in the sample; the detection force (F1=9pN) is held for 5 seconds, and after binding to miR-122, the change in the extension length of the probe molecule compared to the reference length under the detection force is recorded, recorded as LB, as the binding signal intensity of miR-122; the regeneration force (F3=1pN) stays for 10 seconds to completely dissociate the analyte miR-122 bound to the probe, and the probe returns to the level of unbound analyte. initial state.

Step 4: Repeat the mechanical stretching mode in Step 3 to measure the signal intensity of the binding of miR-122 to the probe molecule in two samples of the patient's liver cancer tissue and adjacent tissue.

Step 5: Use the standard curve method to determine the concentration of the analyte miR-122 in the sample. The standard curve in Figure 6A shows that within a specific concentration range, the binding signal intensity is proportional to the concentration of miR-122 molecules in the sample. Correspond the signal intensity measured in step 4 to the standard curve to obtain the quantitative result of miR-122 in the sample, as shown in Figure 6B.

Example 6: Tandem nucleic acid probes with uniform polymerization degree detect and distinguish let-7 family members in serum whose sequences differ only by 1-2 bases: let-7a, let-7b, let-7c and let-7d.

The sequence of let-7a is SEQ ID NO: 10: 5’-UGAGGUAGUAGGUUGUAUAGUU-3’ (RNA);

The sequence of let-7b is SEQ ID NO: 11: 5’-UGAGGUAGUAGGUUGUGUGGUU-3’ (RNA);

The sequence of let-7c is SEQ ID NO: 12: 5’-UGAGGUAGUAGGUUGUAUGGUU-3’ (RNA);

The sequence of let-7d is SEQ ID NO: 13: 5'-AGAGGGUAGUAGGUUGCAUAGUU-3' (RNA).

Specific steps are as follows:

Step 1: According to Example 1, use the hairpin sequence T1 and T2_3, T2_4, T2_5, and T2_6 to synthesize four circular rolling circle amplification templates targeting let-7a, let-7b, let-7c, and let-7d.

The T2_3 sequence is SEQ ID NO:14:

5’-GTCCAGTGCAAGTAGGTTGTATAGTTCTGCTTTTGCAGAACTATACAACCTACTTG CA-3’;

The T2_4 sequence is SEQ ID NO:15:

5’-GTCCAGTGCAAGTAGGTTGTGTGGTTCTGCTTTTGCAGAACCACACAACCTACTTGCA-3’;

The T2_5 sequence is SEQ ID NO:16:

5’-GTCCAGTGCAAGTAGGTTGTATGGTTCTGCTTTTGCAGAACCATACAACCTACTTGCA-3’;

The T2_6 sequence is SEQ ID NO: 17: 5'-GTCCAGTGCAAGTAGGTTGCATAGTTCTGCTTTTGCAGAACTATGCAACCTACTTGCA-3'.

Step 2: Mix the four circular rolling circle amplification templates in equal amounts and dilute them with 3×PBS buffer to a total concentration of 1ng/μL. According to the steps described in Example 2, a tandem nucleic acid probe set that can simultaneously target let-7a, let-7b, let-7c and let-7d and has a uniform degree of polymerization is synthesized in situ.

Step 3: Connect the tandem nucleic acid probe set with a uniform degree of polymerization to magnetic microspheres and place it in a single-molecule magnetic tweezers device. According to the method shown in Figure 7, use known standards of the analyte to sequentially determine the uniform degree of polymerization. The type of each probe in the tandem nucleic acid probe set.

Step 4: Set the mechanical stretching mode of the detection process to: the detection force (F1=6.5pN) is held for 5 seconds, determine the molecular extension length of each probe molecule under the detection force before binding to the test substance, and record it Reference length; the binding force (F2=20pN) stays for 3 minutes, and the target sequence on the probe molecule is exposed under the binding force, identifying and binding the analyte in the sample to be tested; the detection force (F1=6.5pN) stays for 5 seconds , record the change in the extension length of each probe molecule compared to the reference length under the detection force after binding to the analyte, recorded as LB, as the binding signal intensity of the analyte. Since the stability of the analyte binding to non-specific probe molecules is much lower than that of binding to specific probe molecules, under this detection power, the non-specifically bound analyte will dissociate rapidly, leaving only The selection of specifically bound analytes greatly increases the detection specificity of the method, as shown in Figure 8. Renew the force (F3=1pN) and stay for 10 seconds to dissociate all the analytes bound to the probe, and return the probe to the initial state of unbound analytes.

Step 5: Repeat the mechanical stretching mode in Step 4, replace different samples to be tested in sequence, and complete the detection of all samples to be tested. The detection results are shown in Figure 9. The signal intensity generated by the specific probe is much greater than that of the non-specific probe. The signal intensity generated by the opposite sex probe.

Step six: Add 0pM, 1pM, 10pM, 100pM, 1nM and 10nM let-7c to the samples containing 0pM and 10pM let-7b respectively, and then repeat the mechanical stretching mode in step three to verify the strength of let-7c. There is an impact on the specific detection of let-7b. The detection results are shown in Figure 10. The presence of let-7c, which has only one base difference, does not affect the detection specificity of let-7b. However, when the concentration of let-7c is more than 100 times higher than let-7b, it will be affected to a certain extent. affects the detection sensitivity of let-7b.

Example 7: Tandem nucleic acid probes with uniform polymerization degree detect novel coronavirus nucleic acid fragments in saliva and identify virus subtypes. Specific steps are as follows:

Step 1, according to Example 1, use the hairpin structure T1 and T2_7, T2_8, T2_9, T2_10, T2_11 to synthesize five types of novel coronavirus spike genes 614D, 614G, 679N/681P, 679N/681R and 679K/681H. Dotted circular single-stranded rolling circle amplification template.

The sequence of T2_7 is SEQ ID NO:18:

5’-GTCCAGATGCTATCAGGATGTTAACTCTGGTTTTCCAGAGTTAACATCCTGATAGCAT-3’;

The sequence of T2_8 is SEQ ID NO:19:

5’-GTCCAGATGCTATCAGGGTGTTAACTCTGGTTTTTCCAGAGTTAACACCCTGATAGCAT-3’;

The sequence of T2_9 is SEQ ID NO:20:

5’-GTCCAGATGCACTAATTCTCCTCGGTCTGTTTTTCAGACCGAGGAGAATTAGTGCAT-3’;

The sequence of T2_10 is SEQ ID NO:21:

5’-GTCCAGATGCACTAATTCTCGTCGGTCTGTTTTTCAGACCGACGAGAATTAGTGCAT-3’;

The sequence of T2_11 is SEQ ID NO:22:

5’-GTCCAGATGCACTAAGTCTCATCGGTCTGTTTTCAGACCGATGAGACTTAGTGCAT-3’.

Step 2: Mix four circular single-stranded rolling circle amplification templates at equal concentrations and dilute to 1ng/μL with 3×PBS buffer. According to the steps described in Example 2, a tandem nucleic acid probe set that can simultaneously target the five sites of 614D, 614G, 679N/681P, 679N/681R and 679K/681H and has a uniform degree of polymerization is synthesized.

Step 3: The RNA sample of the new coronavirus is fragmented by reacting with fragmentation buffer (New England Biolabs) at 95°C for 10 minutes, and then diluted to a concentration of 5pg/μL with a measurement solution containing 5% saliva.

Step 4: Connect the tandem nucleic acid probe set with a uniform degree of polymerization to magnetic microspheres and place it in a single-molecule magnetic tweezers device. According to the method shown in Figure 7, use known standards of the analyte to sequentially determine the uniform degree of polymerization. The type of each probe in the tandem nucleic acid probe set.

Step 5: Set the mechanical stretching mode of the detection process to: the detection force (F1 = 6pN) is held for 5 seconds, determine the molecular extension length of each probe molecule under the detection force before binding to the test substance, and record it as a benchmark Length; the binding force (F2=20pN) stays for 3 minutes, and the target sequence on the probe molecule is exposed under the binding force, identifying and binding the analyte in the sample; the detection force (F1=6pN) stays for 5 seconds, and the binding time is recorded After detecting the substance, the change in the extension length of each probe molecule compared to the reference length under the detection force is recorded as LB and is used as the binding signal intensity of the substance to be tested. Since the stability of the analyte binding to non-specific probe molecules is much lower than that of binding to specific probe molecules, under this detection power, the non-specifically bound analyte will dissociate rapidly, leaving only The selection of specifically bound analytes greatly increases the detection specificity of the method. Renew the force (F3=1pN) and stay for 10 seconds to dissociate all the analytes bound to the probe, and return the probe to the initial state of unbound analytes.

Step 6: Follow step 3 to measure all the samples to be tested in sequence: negative controls containing only the measurement solution, samples containing wild-type viral RNA fragments, samples containing B.1.617.2 subtype RNA fragments, and samples containing BA.1 subtype RNA. Fragment the sample and record the test results as shown in Figure 11.

Example 8: Connect the single-stranded linear DNA template T3 end-to-end via bridging DNA and T4 DNA ligase to construct a single-stranded circular rolling circle transcription template, as shown in Figure 12. Specific steps are as follows:

Step 1: Design and synthesize single-stranded linear DNA template T3 and bridge DNABridge1.

The sequence of T3 is SEQ ID NO:23:

5’-GTGAGTCGTATTAAGTCAAGTTAACCCTAACCCTAACCCTAACCCTAATCGAGCACGGCTCTACTCTACTCTACTCTACTCTCCTATA-3’;

The sequence of Bridge1 is SEQ ID NO: 24: 5'-TAATACGACTCACTATAGGAGAG-3'.

The 5' end of the single-stranded linear DNA template is phosphorylated, and its sequence contains a promoter sequence and a target sequence. The promoter sequence is the complementary sequence of the T7 promoter sequence, and the target sequence is the complementary sequence of the human telomeric G-quadruplex sequence. The 3’ end sequence of the bridge DNA is complementary to the 5’ end part of the template T3; the 5’ end sequence of the bridge DNA is complementary to the 3’ end part of the template T3.

Step 2: Template T3 and bridging DNA anneal to form a circular structure containing a nick. Template T3 with a concentration of 2 μM and bridge DNA Bridge1 with a concentration of 2 μM were incubated at 75°C for 10 minutes in a buffer containing 10mM Tris-HCl, and then lowered to 20°C at a rate of one degree per minute to perform an annealing reaction.

Step three: Enzyme ligation reaction to synthesize a closed-structure circular rolling circle transcription template. Add T4 DNA ligase and reaction buffer to the annealed product in step 2, incubate at 4°C for 12 hours to perform the enzyme ligation reaction, and then react at 65°C for 10 minutes to inactivate T4 DNA ligase.

Step 4: Purify and recover the closed structure circular rolling circle transcription template. The ligation product in step three is recovered through an ultrafiltration tube, and then treated with DNA exonuclease III and DNA exonuclease I in sequence to digest all linear or nick-containing by-products to obtain a higher purity closed structure. Circular roll transcription template.

Step 5: Polyacrylamide gel electrophoresis detects the products of steps 2, 3 and 4 respectively. The gel concentration was 8%, and the gel was electrophoresed at 100V for 40 minutes, then soaked and stained with the nucleic acid dye Gel-Red, and imaged by the Bio-Rad gel imaging system.

Example 9: Synchronized rolling circle transcription in situ on the flow cell surface to synthesize tandem nucleic acid probes with uniform polymerization degrees, as shown in Figure 13. Specific steps are as follows:

Step 1: Preparation of the flow cell with digoxigenin antibody coupled to the inner surface. The preparation of the flow cell and the modification of the digoxigenin antibody on the inner surface were carried out according to the classic experimental procedures for single-molecule experiments.

Step 2: anneal the complementary sequence T7_C of the promoter sequence to the circular single-stranded rolling circle transcription template to form a circular rolling circle transcription template in which part of the promoter sequence is a double-stranded structure and the other part is a single-stranded structure.

The sequence of the complementary sequence T7_C of the initiating sequence is SEQ ID NO: 25: 5'-TAATACGACTCACTATAGG-3'.

Step 3: Form a rolling circle transcription complex that can initiate synchronously. Configure a transcription system containing defective substrates (UTP, ATP and CTP), where the UTP in the defective substrate is digoxin-modified UTP. The concentration of the rolling circle transcription template in the transcription system is 1ng/μL. Incubate at 37°C for 5 minutes to allow T7 RNA polymerase to fully recognize the initiation sequence of the double-stranded structure, form a rolling circle transcription complex, initiate transcription, and then pause when it first encounters the base C site on the template. Use an ultrafiltration tube to recover the paused rolling circle transcription complex.

Step 4: Add the recovered rolling circle transcription complex in Step 3 to a flow cell with digoxigenin antibody coupled to the inner surface, and react at room temperature for 10 minutes to utilize the digoxigenin-labeled nascent transcripts. Affinity interactions of digoxin and digoxin antibodies are coupled to the inner surface of the flow cell.

Step 5: Add complete reaction substrate NTPmix, restart the transcription process, and continue incubating at 37°C for 10 minutes.

Step 6: Prepare a reaction substrate mixed with biotin-labeled UTP. The ratio of biotin-labeled UTP to upper UTP is equal to 1:9. Add it to the flow cell and continue to incubate at 37°C for 1 minute so that rolling circle transcription synthesizes polymers with a uniform degree of polymerization. The 3' end of the tandem nucleic acid probe is labeled with biotin.

Step 7: Add 30mMEDTA to terminate the transcription reaction.

Step 8: Anneal spacer sequence complementary sequence 2: Flank2.

The sequence of Flank2 is SEQ ID NO:26:

5’-CTTGACTTAATACGACTCACTATAGGAGAGTAGAGTAGAGTAGAGTAGAGCCGTG CTCG-3’.

Adding Flank2 at a concentration of 100nM makes the non-functional spacer sequence in the tandem nucleic acid probe with a uniform degree of polymerization form a double-stranded structure.

Step 9: Connect newly synthesized tandem nucleic acid probes with uniform polymerization degree to magnetic microspheres. The magnetic microsphere M-270 is diluted 100 times with 3×PBS buffer and then added to the flow cell. The surface of the magnetic microsphere M-270 is modified with streptavidin, which can interact with the biotin labeled at the 3' end of the tandem nucleic acid probe. effect.

Example 10: Uniform polymerization tandem nucleic acid probes prepared by rolling circle transcription were used to detect the effect of small molecule ligand PDS on the structural stability of RNAG quadruplex. Specific steps are as follows:

Step 1: According to the steps described in Example 8, linear single-stranded DNA template T3 and bridging DNABridge1 are used to synthesize a circular rolling circle transcription template containing the complementary sequence of the G-quadruplex specific structure sequence.

Step 2: Synchronize in-situ synthesis of tandem nucleic acid probes that can form a specific RNAG quadruplex structure and have a uniform degree of polymerization according to the steps described in Example 9.

Step 3: Connect the tandem nucleic acid probes with a uniform degree of polymerization to magnetic microspheres and place them in the single-molecule magnetic tweezers device. Set the mechanical stretching mode of the detection process, as shown in Figure 14: Regeneration force (F3=50pN) stays 20 seconds to unfold all the G-quadruplex specific structures on the probe; the detection force (F1=20pN) stays for 10 seconds to determine the molecular extension length of the probe under the detection force before the G-quadruplex structure is formed, and Record as the reference length; the binding force (F2=2pN) stays for 60 seconds. Under the binding force, all target sequences on the probe molecule are folded into a G-quadruplex specific structure, and the analyte in the sample to be tested is recognized and combined at the same time. , such as small molecule ligand PDS; the detection force (F1=20pN) is stayed for 60 seconds, and the change in the extension length of the probe molecule compared to the reference length under the detection force after binding to the analyte is recorded, recorded as LB, as Determine the unfolding probability of the G-quadruplex specific structure and determine the binding signal intensity of the test substance; stay with the regeneration force (F3=50pN) for 20 seconds to fully unfold the G-quadruplex-specific structure on the probe.

Step 4: Repeat the mechanical stretching mode in step 3, and use the measurement solution (100mM potassium chloride) as the control group to determine that the small molecule ligand PDS can significantly increase the RNAG quadruplex structure formed by human telomeric repeat sequences. Stability, as shown in Figure 15.

Claims (10)

1. A molecular detection method, characterized in that: using multi-channel single molecule mechanics technology to simultaneously manipulate multiple molecular probes to detect the analyte; both ends of the molecular probe are connected to the carrier through labeled coupling functional groups; The carrier is used to exert pulling force on the molecular probe. 2. The method according to claim 1, characterized in that: the molecular probe is a tandem nucleic acid probe, and the monomer of the tandem nucleic acid probe contains a target sequence; under a certain range of pulling force, the target sequence can form Binds to the specific structure of the analyte. 3. The method according to claim 2, characterized in that the tandem nucleic acid probe is prepared by rolling circle amplification or rolling circle transcription. 4. The method according to claim 3, characterized in that: the rolling circle amplification includes the following steps: (i) Initiation of rolling circle amplification: rolling circle amplification primers, DNA polymerase and rolling circle amplification template are required; the rolling circle amplification template contains the promoter sequence and the complementary sequence of the target sequence; the promoter sequence can be combined with the rolling circle amplification primer; (ii) Extension of rolling circle amplification: all necessary nucleotides are used as substrates and amplification is continued for a period of time according to the required degree of polymerization of the tandem nucleic acid probes. 5. The method according to claim 3, characterized in that: the rolling ring transcription includes the following steps: (i) Initiation of rolling circle transcription: RNA polymerase and rolling circle transcription template are required; the rolling circle transcription template contains the promoter sequence and the complementary sequence of the target sequence; the promoter sequence is a sequence that binds RNA polymerase; in this step, the newly generated RNA transcripts are labeled with coupling functional groups; (ii) Elongation of rolling circle transcription: all necessary nucleotides are used as substrates and transcription is continued for a period of time based on the desired degree of polymerization of the tandem nucleic acid probe. 6. The method according to claim 4 or 5, characterized in that: the extending step is performed while being fixed on a carrier. 7. The method according to claim 3, characterized in that: rolling circle amplification or rolling circle transcription includes a process of pausing and then restarting. 8. The method according to claim 7, characterized in that: the reaction reagents for rolling circle amplification or rolling circle transcription lack at least one of the necessary nucleotide substrates, and the polymerase needs to use the missing nucleoside when working. Pause on acid substrate. 9. The method according to any one of claims 1 to 5, 7, and 8, characterized in that: the detection process using multi-channel single molecule mechanics technology includes binding force and detection force: under the binding force, the target sequence Form a specific structure that can bind to the analyte; detect the analyte under detection force; When the detection force is a constant force, record the length of the tandem nucleic acid probe when there is no analyte as the reference length. After changing from the binding force to the detection force, calculate the difference between the length of the tandem nucleic acid probe and the reference length as the signal intensity. When the detection force is a gradient force, record the length change curve of the tandem nucleic acid probe when there is no analyte as the reference curve. During the sample detection process, the difference between the length change curve of the tandem nucleic acid probe and the reference curve is calculated as the signal intensity under the detection force; According to the signal intensity, the presence of the analyte in the sample, the concentration of the analyte, the binding strength and binding speed of the analyte to the specific structure are detected. 10. The method according to any one of claims 1 to 5, 7, and 8, characterized in that: simultaneously operating a plurality of tandem nucleic acid probes in a group, and the target sequences of various tandem nucleic acid probes in the group are for identification. Sequence polymorphism.