Split aptamer probe set and preparation method thereof, kit and protein detection method

Abstract

In order to solve the problem that the complete long-chain aptamer has a secondary structure, which leads to nonspecific and false positive signals, the present invention provides a split aptamer probe group and its preparation method, a kit and a protein detection method based on the click chemistry synthesis method. The split aptamer probe group is a pair of split aptamers S1 and S2 that can highly specifically identify the target protein, which are obtained by splitting at a specific site of the complete aptamer. The present invention realizes the protein template split aptamer connection by click chemistry, converts the binding event of the protein and the split aptamer into the output of the connected nucleic acid product, and realizes multifunctional signal transduction such as electrochemistry, fluorescence, and colorimetry. The kit prepared by this method does not need to be stored and transported at low temperature, and the reaction does not need to be heated. It can react efficiently at room temperature. The protein is detected by the recognition of the target protein-specific split aptamer and the click chemistry reaction, which has the characteristics of low background and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and in particular relates to a split aptamer probe group and a preparation method thereof, a kit and a protein detection method. Background Art

[0002] Proteins are important macromolecules that make up everything in the body, are basic organic substances that make up cells, and are the main executors of various life functions. At the same time, proteins are also the main target molecules for drugs, pathogenic factors, etc. to act on the body, so the detection of some specific proteins has become a favorable means of medical diagnosis. In normal cells, the values ​​of disease-related proteins are very low or even undetectable, but they are expressed in some malignant tumors. As biomarkers for cancer diagnosis, rapid and sensitive detection of protein disease markers is beneficial to the diagnosis, treatment and prognosis of cancer. At present, the main detection technologies for proteins include affinity chromatography, enzyme-linked immunosorbent assay, protein imprinting technology, immunohistochemistry technology, etc. Most of these methods are established by using antibody-antigen reactions. However, the preparation of antibodies for some proteins is difficult, and the poor stability of antibodies, temperature sensitivity, and difficulty in achieving site-specific modification have limited the rapid development of antibodies in protein research.

[0003] As a new type of protein recognition probe, aptamers can overcome the shortcomings of antibodies and provide a sensitive and specific detection mode for protein detection. Due to the strong affinity and high selectivity of nucleic acid aptamers to target molecules, as well as the advantages of easy synthesis, easy modification, and reusability, aptamers can be used as recognition elements to replace traditional antibodies in biological analysis systems. However, complete aptamers have problems such as nonspecific adsorption or false positives.

[0004] The aptamer is split into two fragments, and the nucleotides in the binding region remain intact. When the target is present, the local effective concentration will increase, inducing the reassembly of the split aptamer; but in the absence of the target, the split aptamer is in a low-concentration free state, which can avoid nonspecific and false-positive signals and improve the signal-to-noise ratio. However, it may be accompanied by a decrease in the binding affinity between the aptamer and the target, resulting in a decrease in the true signal and inconvenience in detection. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a split aptamer probe group, a preparation method, a kit and a protein detection method based on a click chemistry synthesis method.

[0006] Click chemistry is a new type of chemical synthesis method that synthesizes macromolecules with specific functions through efficient and rapid reactions between small molecules. In the fields of biology and medicine, click chemistry has been widely used in bioimaging, drug discovery, and gene regulation. However, there are still no reports on the application of click chemistry to protein template split aptamer ligation reactions to achieve protein detection. This method can be carried out at room temperature and under enzyme-free conditions, which will provide new tools for bioanalysis and medical diagnosis. At the same time, for the detection scheme of this method, combining split aptamers can achieve simple, rapid, and low-cost signal transduction, while greatly increasing sensitivity and specificity, which is conducive to the widespread promotion of such protein template split aptamer ligation reactions based on click chemistry in practical applications.

[0007] The present invention first proposes a split aptamer probe set, wherein the probe set is a pair of split aptamers S1 and S2 that can recognize the target protein with high specificity, and is obtained by splitting at specific sites of the complete aptamer.

[0008] Furthermore, the 3' end of the split aptamer S1 is azide-modified, the 5' end of S2 is alkyne-modified, and the base lengths of S1 and S2 are 10-50 nt, preferably 10-25 nt.

[0009] Furthermore, the 5' end of the split aptamer S1 and the 3' end of S2 are modified with detection groups for multifunctional signal transduction output, specifically any one of electrochemical, fluorescent, colorimetric or lateral flow chromatography groups, including but not limited to thiol-methylene blue (SH-MB), fluorescent-quenching group (FAM-Dabcyl), split DNAzyme fragment, etc., for electrochemical, fluorescent, colorimetric and visual detection.

[0010] Preferably, the detection group is a DNAzyme fragment that splits the aptazyme.

[0011] The present invention firstly proposes a method for preparing a split aptamer probe group, which specifically comprises: selecting a loop region of an aptamer away from a binding site for splitting, screening the split aptamer by sequence fitting to determine the split site; and performing sequence synthesis according to the split site to obtain a split aptamer probe group.

[0012] The present invention also provides a split aptamer probe group kit, comprising the split aptamer probe group, for protein detection.

[0013] Finally, the present invention proposes a split aptamer probe group protein detection method, specifically: adding the target protein to a buffer solution containing split aptamer probe groups S1 and S2, using the target protein as a template to induce the split aptamers S1 and S2 to approach each other, and generating a detection signal.

[0014] Furthermore, the split aptamer probe set has a concentration of 20-500 nM.

[0015] Furthermore, when the split aptamer probe set with the 3' end of S1 modified with azide and the 5' end of S2 modified with alkyne is used for detection, a ligation reaction occurs, specifically a copper-catalyzed azide-alkyne cycloaddition reaction or a stress-promoted azide-alkyne cycloaddition reaction.

[0016] Furthermore, the copper-catalyzed azide-alkyne cycloaddition reaction requires the addition of a catalyst, specifically any one of a monovalent copper catalyst or copper nanoparticles.

[0017] Beneficial effects:

[0018] The present invention adopts split aptamers to replace traditional antibody recognition elements, realizes protein template split aptamer connection by click chemistry, converts the binding event of protein and split aptamer into the output of connecting nucleic acid products, and realizes electrochemical, fluorescent, colorimetric and other multifunctional signal transduction. The kit prepared by the method does not need low temperature storage and transportation, and the reaction does not need heating, and can react efficiently at room temperature. The method realizes the detection of protein through the recognition of target protein-specific split aptamers and click chemical reaction, and has the characteristics of low background and high sensitivity; and combined with split aptamer enzymes, the recognition probe and the signal output unit can be directly integrated, without additional probe modification and instrument detection, and naked eye visualization detection can be realized; in addition, the detection method also has the advantages of low cost and simple and convenient operation, and is suitable for rapid detection of disease markers in some areas lacking central laboratories and professionals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of a split aptamer probe set, preparation method, and kit for protein detection. DETAILED DESCRIPTION

[0020] A split aptamer probe group, characterized in that the probe group is a pair of split aptamers S1 and S2 that can highly specifically recognize a target protein, and is obtained by splitting at a specific site of a complete aptamer; optionally, the 3' end of the split aptamer S1 is azide-modified, and the 5' end of S2 is alkyne-modified, and the base lengths of S1 and S2 are 10-50 nt, preferably 10-25 nt; the 5' end of the split aptamer S1 and the 3' end of S2 are modified with a detection group for multifunctional signal transduction output, specifically any one of an electrochemical, fluorescent, colorimetric or lateral flow chromatography group, including but not limited to thiol-methylene blue (SH-MB), a fluorescent-quenching group (FAM-Dabcyl), a split DNAzyme fragment, etc., for electrochemical, fluorescent, colorimetric and visual detection; preferably, the detection group is a DNAzyme fragment of a split aptamer enzyme.

[0021] When the 3' end of the split aptamer S1 is modified with azide and the 5' end of S2 is modified with alkyne, the protein template can induce S1 and S2 to approach each other, and the azide and alkyne groups undergo a click connection reaction, thereby achieving stable signal output; if the 3' end of the split aptamer S1 and the 5' end of S2 are not modified, the protein template can also induce S1 and S2 to approach each other and generate signals, but the signals are weaker than those generated by the click connection reaction caused by modification.

[0022] The method for preparing the above split aptamer probe set is specifically as follows: selecting the loop region of the aptamer away from the binding site for splitting, screening the split aptamer by sequence fitting to determine the split site; and performing sequence synthesis according to the split site to obtain the split aptamer probe set.

[0023] A split aptamer probe group kit comprises the split aptamer probe group and is used for protein detection.

[0024] A split aptamer probe group protein detection method, specifically: adding a target protein to a buffer solution containing split aptamer probe groups S1 and S2, using the target protein as a template, inducing the split aptamers S1 and S2 to approach each other, and generating a detection signal; the split aptamer probe group concentration is 20-500nM; when using the split aptamer probe group with the 3' end of S1 modified by azide and the 5' end of S2 modified by alkyne for detection, a connection reaction will occur, specifically a copper-catalyzed azide-alkyne cycloaddition reaction or a stress-promoted azide-alkyne cycloaddition reaction; the copper-catalyzed azide-alkyne cycloaddition reaction requires the addition of a catalyst, specifically any one of a monovalent copper catalyst or copper nanoparticles.

[0025] The present invention is applicable to any splittable aptamer-protein interaction system, such as VEGF 165 , PDGF-BB, SARS-CoV-2 protein, etc., whose aptamers all contain a cleavable stem-loop structure. Examples 1 to 9 are specifically developed with PDGF-BB protein containing a typical three-way stem-loop structure as the target protein.

[0026] Examples 1 to 3 are specific examples of a split aptamer probe set and a preparation method thereof.

[0027] Example 1

[0028] The loop region I of the aptamer far from the binding site was selected for splitting, and the split aptamers were screened by sequence fitting to determine the split site; the sequence was synthesized according to the split site to obtain a split aptamer probe set. The probe set is a pair of split aptamers S1 and S2 that can highly specifically recognize the target protein. The 3' end of S1 is azide-modified, and the 5' end of S2 is alkyne-modified. The 5' end of S1 and the 3' end of S2 are modified with detection groups for multifunctional signal transduction output. The base lengths of S1 and S2 are 10nt and 25nt.

[0029] Example 2

[0030] The loop region II of the aptamer far from the binding site is selected for splitting, and the split aptamers are screened by sequence fitting to determine the split site; the sequence is synthesized according to the split site to obtain a split aptamer probe set. The probe set is a pair of split aptamers S1 and S2 that can highly specifically recognize the target protein. The 5' end of S1 and the 3' end of S2 are modified with detection groups for multifunctional signal transduction output. The 3' end of S1 is azide-modified, and the 5' end of S2 is alkyne-modified. The base lengths of S1 and S2 are 13nt and 22nt.

[0031] Example 3

[0032] The loop region III of the aptamer close to the binding site is selected for splitting, and the split aptamers are screened by sequence fitting to determine the split site; the sequence is synthesized according to the split site to obtain a split aptamer probe set. The probe set is a pair of split aptamers S1 and S2 that can highly specifically recognize the target protein. The 5' end of S1 and the 3' end of S2 are modified with detection groups for multifunctional signal transduction output. The 3' end of S1 is azide-modified, and the 5' end of S2 is alkyne-modified. The base lengths of S1 and S2 are 17nt and 18nt.

[0033] Examples 4 to 6 are specific examples of a split aptamer probe set kit.

[0034] Example 4

[0035] A kit was prepared using the probe set prepared in Example 1, and a pair of 200 nM split aptamer probe sets, including split aptamer S1 with SH and N3 modified at both ends, were added. E , split aptamer S2 modified with DBCO and MB E Mix, add a quantitative amount of target protein, incubate at room temperature for 0.5 hours, mix in a total volume of 100 μL of Phys2 buffer (pH 7.4), and react at room temperature for 1 hour.

[0036] Example 5

[0037] A kit was prepared using the probe set prepared in Example 2, and a pair of 100 nM split aptamer probe sets, including split aptamer S1 with Dabcyl and N3 modified at both ends, were added. F , split aptamer S2 modified with DBCO and FAM F Mix, add a quantitative amount of target protein, incubate at room temperature for 0.5 hours, mix in a total volume of 300 μL of PBS buffer (pH 7.4), and react at room temperature for 1 hour.

[0038] Example 6

[0039] A kit was prepared using the probe set prepared in Example 3, and a pair of 100 nM split aptamer probe sets, including split aptamer S1 with split DNAzyme fragments and N3 modified at both ends, were added. C , split aptamer S2 modified with DBCO and split DNAzyme fragment C After mixing, a quantitative amount of target protein was added and incubated at room temperature for 0.5 h. A colorimetric agent was added and mixed in a total volume of 300 μL of HEPES buffer (pH 7.4) and reacted at room temperature for 1 h.

[0040] Examples 7 to 9 are specific examples of the testing method.

[0041] Example 7

[0042] The detection method performed using the kit of Example 4 uses the target protein as a template to induce the splitting of the aptamer S1 E and S2 E Close to the connection, an electrochemical detection signal is generated. The electrochemical measurement was performed on a CHI 1040C electrochemical workstation using a typical three-electrode electrochemical system configuration at room temperature. Ag / AgCl (3.0M KCl) electrode and platinum (Pt) wire were used as reference electrode and counter electrode, respectively. A commercially available gold disk electrode (2 mm in diameter) was used as the working electrode. A gold-modified screen-printed electrode can also be used as a signal output element. Alternating current voltammetry (ACV) with a frequency of 10 Hz and an amplitude of 25 mV was used in a Phys2 buffer electrolyte. Before sensor fabrication, the gold electrode was first cleaned by polishing with 0.05 μm alumina slurry, then ultrasonically treated with water and ethanol, and then electrochemically cleaned in 0.05-0.5 M H2SO4 until a characteristic cyclic voltammetry (CV) curve was obtained. The protein template split aptamer connection reaction proposed in the present invention does not require the use of enzymes and catalysts, is simple to operate, and only requires a pair of split aptamers and target proteins to be mixed at room temperature. The detection limit of this detection scheme is as low as 1 pM. The relationship between electrochemical detection of target protein and signal gain is shown in Table 1.

[0043] Table 1 Relationship between electrochemical detection of target protein and signal gain

[0044]

[0045] Example 8

[0046] The detection method performed using the kit of Example 5 uses the target protein as a template to induce the splitting of the aptamer S1 F and S2 F Close to the connection, a fluorescence detection signal is generated. The FAM fluorescence intensity from 500nm to 600nm with an excitation wavelength of 488nm was recorded using an FL6500 fluorescence spectrophotometer (Perkin Elmer, Inc., USA), and the excitation wavelength was 526nm. The protein template binds to the split aptamer, restoring the three-way stem-loop structure of the complete aptamer, bringing the Dabcyl-FAM group close to achieve a significant decrease in the fluorescence signal. The detection limit of the target protein of this detection scheme is as low as 15pM.

[0047] Example 9

[0048] The detection method performed using the kit of Example 6, wherein the target protein is used as a template to induce the splitting of the aptamer S1 C and S2 C Close connection generates colorimetric signals. This detection scheme does not require the use of any detection instruments and can be directly visualized by the naked eye, greatly saving costs and time.

[0049] Table 2 shows all nucleic acid sequences used in the present invention:

[0050] Table 2 All nucleic acid sequences used in the present invention

[0051]

[0052]

[0053] The above contents are only preferred embodiments of the present invention and are not intended to limit the implementation scheme of the present invention. A person skilled in the art can easily make corresponding changes or modifications based on the main concept and spirit of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope required by the claims.

Claims

1. A method for detecting a protein using a split aptamer probe set: adding a target protein to a buffer solution containing split aptamer probe sets S1 and S2, using the target protein as a template to induce the split aptamers S1 and S2 to approach each other, and generating a detection signal; the probe set used is a pair of split aptamers S1 and S2 that can highly specifically recognize the target protein, and is obtained by splitting the loop region of the intact aptamer; the 3' end of the split aptamer S1 is azide-modified, and the 5' end of S1 is: 5'- TGGG -3'; the 5' end of S2 is alkyne-modified, and the 3' end of the split aptamer S2 is: 5'- TTTGGGAGGGAGGGT -3'; the base lengths of S1 and S2 are 25-50 nt; a split aptamer probe set is used, and a ligation reaction occurs during detection, specifically a copper-catalyzed azide-alkyne cycloaddition reaction or a stress-promoted azide-alkyne cycloaddition reaction.

2. The method according to claim 1, characterized in that The split aptamer probe set concentration is 20-500 nM.

3. The method according to claim 1, characterized in that The copper-catalyzed azide-alkyne cycloaddition reaction adds a catalyst, specifically any one of a monovalent copper catalyst or copper nanoparticles.

4. A method for preparing a split aptamer probe group used in the method for detecting proteins using a split aptamer probe group as described in any one of claims 1 to 3, specifically comprising: selecting a loop region of the aptamer away from the binding site for splitting, screening the split aptamer by sequence fitting to determine the split site; and performing sequence synthesis according to the split site to obtain a split aptamer probe group.

5. A kit prepared by using the split aptamer probe set used in the method for detecting protein using the split aptamer probe set according to any one of claims 1 to 3, characterized in that: The probe set comprising claim 1.

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