Enzymatic DNA in-situ synthesis method of antibody coupled oligonucleotide

Abstract

The invention discloses an antibody coupling oligonucleotide enzymatic DNA in-situ synthesis method. The invention particularly relates to an antibody coupling oligonucleotide enzymatic DNA in-situ synthesis method in the technical field of enzymes. The in-situ synthesis method of the antibody-coupled oligonucleotide comprises the following steps: 1) oligonucleotide synthesis: synthesizing an oligonucleotide chain by a chemical method; 2) coupling of an antibody and oligonucleotide: coupling the oligonucleotide obtained in the step 1) with the antibody through a covalent bond to form a compound; and 3) TdT enzymatic ssDNA in-situ synthesis: taking a deoxyribonucleoside triphosphate monomer as a raw material, and adopting TdT enzyme to extend at the oligonucleotide 3 '-OH end of the compound to obtain an enzymatic ssDNA in-situ synthesis product. The antibody is combined with the TDT enzyme, so that enzymatic DNA synthesis with the antibody as a carrier is realized, the in-situ synthesis efficiency and stability of the antibody-coupled oligonucleotide are improved, and the method has an application prospect.

Description

Technical Field

[0001] The present invention specifically relates to an antibody-coupled oligonucleotide enzymatic DNA in situ synthesis method in the field of enzyme technology. Background Art

[0002] Currently, in the field of DNA synthesis, chemical synthesis using solid-phase phosphoramidite technology is still the mainstream synthesis technology. The maximum synthesis length of this method is 200-300nt, its synthesis efficiency is unstable and it produces toxic waste. Compared with chemical synthesis, enzymatic synthesis allows reactions to be carried out in an aqueous environment, its reaction conditions are milder, and no toxic compounds are required, making it possible to synthesize DNA anywhere, which is expected to solve many problems existing in chemical synthesis.

[0003] Terminal deoxynucleotidyl transferase (TdT) was first isolated from calf thymus. Its molecular weight is about 60 kDa. In the 1980s, it was confirmed that it plays a role in the V(D)J recombination of immune system antibodies and randomly synthesizes antibody variable sequence regions. TdT is a template-independent single-stranded DNA polymerase with rapid polymerization activity that is template-independent. The entire catalytic process does not require denaturation, renaturation, and extension reactions. With an oligonucleotide as a primer, it catalyzes the polymerization of deoxyribonucleoside triphosphates (dNTPs) to the 3' end of the oligonucleotide under constant temperature and metal ion-assisted conditions to achieve long-chain DNA synthesis. Importantly, TdT has low selectivity for substrates and has a fast catalytic reaction rate for all dNTPs. Any dNTP, ribonucleoside triphosphate (rNTP), and modified nucleoside triphosphate analogs (such as fluorescently modified dNTPs, biotin-modified dNTPs, artificial bases, etc.) can be used as its substrates. In addition to enzymatic DNA synthesis, TdT can also be used as a medium for identifying and amplifying signals and is widely used in the construction of biosensors for the detection of nucleases, exosomes, proteins, and microRNAs.

[0004] When TdT performs enzymatic DNA synthesis on a solid phase carrier, the 5' end of the nucleotide chain is covalently or non-covalently attached to the solid support, exposing the 3' end of the nucleotide chain, laying the foundation for enzymatic synthesis. Solid-phase gene chips can achieve rapid detection and analysis of a variety of biological molecules such as DNA, RNA, and proteins by fixing oligonucleotide probes on the surface of the chip. In TDT enzymatic DNA synthesis, this technology can be used to synthesize DNA of a specific sequence, perform local synthesis reactions through reaction sites on the solid phase chip, and then assemble into long-chain DNA molecules. In addition to solid phase carriers, TdT can also directly synthesize oligonucleotides on magnetic beads, cells, or biological molecules.

[0005] Antibody-Oligonucleotide Conjugates (AOC) are new synthetic bioconjugates composed of monoclonal antibodies and oligonucleotides, in which the antibody part can specifically target specific cell surface antigens, and the oligonucleotide can regulate the expression of target genes. The characteristics of AOC combined with antibodies and oligonucleotides are widely used in modern biotechnology and drug research and development.

[0006] TDT enzyme can perform in situ DNA synthesis in a template-independent manner. If oligonucleotide sequences can be synthesized on the oligo chain of AOC, the presence of antigen signals in the original sample can be determined by detecting the presence of the synthesized sequence, which provides a new idea for antigen / antibody detection and has broad application prospects. Summary of the invention

[0007] The main problem to be solved by the present invention is how to prepare antibody-coupled oligonucleotides without relying on a template.

[0008] In order to solve the above problems, the present invention provides a method for in situ synthesis of antibody-coupled oligonucleotides.

[0009] The in situ synthesis method of antibody-coupled oligonucleotide provided by the present invention comprises the following steps: 1) Oligonucleotide synthesis: chemical synthesis of oligonucleotide chains; 2) Conjugating the antibody and oligonucleotide: conjugating the oligonucleotide in step 1) to the antibody via a covalent bond; 3) TdT enzymatic in situ ssDNA synthesis: Using deoxyribonucleoside triphosphate monomers as raw materials, TdT enzyme is used to extend the 3'-OH end of the oligonucleotide to obtain the enzymatic in situ ssDNA synthesis product.

[0010] In the above method, the coupling in step 2) is performed using an oligonucleotide coupling kit (Hangzhou Lewei Biomedical Technology Co., Ltd., catalog number LW-AOC-010).

[0011] In the above method, the TdT enzyme in step 3) can add nucleotides to the end of the DNA chain, plays an important role in the replication and repair of DNA, and participates in important physiological processes such as V(D)J recombination and DNA repair in vivo.

[0012] In this article, due to the low selectivity of TDT enzyme for substrates, fluorescently modified and electrochemically modified nucleotides can be used as substrates for in situ synthesis of AOC. After the in situ synthesis product synthesized using chemically modified dNTP as raw material binds to the target antigen, an electrochemiluminescence instrument can be used for fluorescence intensity detection, which is helpful for the localization and quantitative analysis of target proteins in cells or tissues.

[0013] In the above method, the dNTP used in the ssDNA in situ synthesis in step 3) may be a chemically modified dNTP.

[0014] Furthermore, the chemically modified group is located at the -NH2 group on the bases A, G, C, and T.

[0015] In the above method, the ssDNA in situ synthesis reaction system in step 3) is: 6 μL antibody (final concentration is 2 ng / mL), 3 μL 10×Tris-HCl (final concentration is 0.1 M), 3 μL CoCl2 (final concentration is 1mM), 3μL Triton (final concentration is 0.1%), 3μL dNTP (final concentration is 0.5mM), 3μL TdT (final concentration is 0.5mg / mL), and ddH2O is added to 30μL.

[0016] In the above method, the in-situ synthesis reaction conditions described in step 3) are: 30°C, 10 min; 98°C, 2 min.

[0017] In the above method, the step of coupling the antibody and oligonucleotide in step 2) is as follows: 1) activating the oligonucleotide; 2) purifying the activated oligonucleotide to obtain purified oligonucleotide; 3) activating the antibody; 4) desalting and purifying the antibody to obtain a purified antibody; 5) Antibody-oligonucleotide coupling: The purified antibody obtained in step 4) is coupled with the oligonucleotide obtained in step 2) at a molar ratio of 1:5, and the reaction is carried out at 4° C. in the dark for 12 hours to obtain a complex, which is an antibody conjugate coupled with the oligonucleotide.

[0018] In the antibody conjugate, the 5' end of the oligonucleotide is linked to the antibody.

[0019] The present invention also provides an in-situ synthesis product prepared by the method described above.

[0020] The present invention also provides the use of TdT enzyme in antibody-coupled oligonucleotide enzymatic ssDNA in situ synthesis.

[0021] The present invention also provides the application of the above-mentioned in situ synthesis method in the in situ synthesis of antibody-coupled oligonucleotide enzymatic ssDNA.

[0022] The application of the above-mentioned in situ synthesis method in enhancing the recognition ability of antibodies also falls within the scope of protection claimed by the present invention.

[0023] The present invention also provides the use of the above-mentioned synthetic product in enhancing the recognition ability of antibodies.

[0024] The present invention proposes an innovative biomaterial stationary phase - antibody, which forms a complex by connecting the 5' end of the oligonucleotide to the antibody, and uses the TdT enzyme to perform in situ synthesis at the 3' end of the oligonucleotide of the complex. The antibody that specifically binds to the target protein serves as a new synthetic carrier for TdT synthesis, and through its specific recognition ability, it provides a new strategy for amplifying biological signals.

[0025] The present invention improves the in situ synthesis efficiency and stability of antibody-coupled oligonucleotides by combining an antibody carrier with TDT enzyme amplification, and uses TdT enzyme to catalyze chemically modified dNTPs for DNA synthesis, which makes it possible for the amplification signal of the TDT enzyme to be output in an electrochemical form.

[0026] The process flow proposed by the present invention has the following advantages: 1) High efficiency of enzymatic reaction: The present invention performs enzymatic in situ ssDNA synthesis based on the existing antibody-nucleic acid coupling technology. The TdT enzyme used is a template-independent synthetase. The enzyme can complete the extension of ssDNA under constant temperature conditions, does not require a complex thermal cycle system, and reduces the number of synthesis steps; 2) High throughput: The process steps of the present invention are simple, and the process does not require a complex thermal cycle system, which can achieve automation and throughput; 3) Good specificity: The synthetic antibody-coupled oligonucleotides are targeted, and the antibodies can be directly labeled for the detection of target substrates by modifying the oligonucleotides to a certain extent; 4) Wide application scenarios: Specific target sequences can be customized and synthesized based on AOC according to customer research needs, which is suitable for various biological research and clinical application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the process flow of the present invention.

[0028] Figure 2 The figure is the electrophoresis detection result of TdT enzyme synthesis products. Lane 1 is DNA Ladder, Lane 2 is the product of enzymatic synthesis using dNTP as substrate on 2ng / mL oligonucleotide-coupled antibody; Lane 3 is the product of enzymatic synthesis using chemically modified dNTP as substrate on 2ng / mL oligonucleotide-coupled antibody; Lane 4 is the product of enzymatic synthesis using dATP as substrate on 2ng / mL oligonucleotide-coupled antibody; Lane 5 is the product of enzymatic synthesis using chemically modified dATP as substrate on 2ng / mL oligonucleotide-coupled antibody. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0030] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0031] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0032] The recombinant human interleukin IL-12β antibody in the following examples was purchased from Beijing Biolab Technology Co., Ltd., catalog number: JN0401.

[0033] The Terminal Deoxynucleotidyl Transferase TdT (2.5 mg / mL) in the following examples was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 10302ES76.

[0034] The dNTPs in the following examples were purchased from Novazon Biotechnology, item number P031-01; dATP was purchased from Novazon Biotechnology, item number P034-01; chemically modified dNTPs were purchased from Xi'an Qiyue Biotechnology Co., Ltd., item number Q-0160879; and chemically modified dATP was purchased from Baiying Biotechnology, item number 17038.

[0035] Example 1. Preparation and detection of antibody-conjugated oligonucleotides The present invention connects antibodies and oligonucleotide chains, and based on them, uses four substrates, namely, chemiluminescent modified dATP, chemiluminescent modified dNTP, natural dATP, and natural dNTP as raw materials for enzymatic DNA synthesis. Among them, the chemiluminescent modification group is located at the -NH2 group on the A, G, C, and T bases. After the TDT enzyme synthesis reaction is completed, the single-stranded nucleotide chain is separated, and the quality inspection is performed using a denatured polyacrylamide gel, and a chemiluminometer is used to detect whether the chemiluminescent modified dATP and dNTP are successfully utilized during the DNA synthesis process.

[0036] 1. Antibody-oligonucleotide conjugation The interleukin antibody IL-12β was prepared by chemically synthesizing a 20 nt oligonucleotide fragment (nucleotide sequence: 5'-GCTCGGTACCTCGCGAATGC-3', SEQ ID No: 1), and the control antibody and control oligonucleotide in the oligonucleotide coupling kit (Hangzhou Lewei Biomedical Technology Co., Ltd., catalog number LW-AOC-010) were used to perform coupling according to the steps described in the instruction manual: 1) Oligonucleotide activation: Take 10 μL oligonucleotide, add 2 μL oligonucleotide activator, add 2 μL buffer A, 10 μL buffer B, shake and mix, centrifuge briefly, and incubate in the dark for 2 hours; 2) purifying the activated oligonucleotide to remove unbound activating agent to obtain purified oligonucleotide; 3) Antibody activation: Take out 6 μL of antibody (interleukin antibody IL-12 or control antibody), add 4 μL of buffer C and 1 μL of antibody activator, shake and mix, centrifuge briefly, and incubate at room temperature in the dark for 1 hour; 4) Antibody desalting and purification: Add 50 μL of PBS to the antibody activation system in step 3), mix well and perform desalting. After desalting, transfer the collected antibodies to a 0.2 mL EP tube again, shake and mix well, and centrifuge to obtain the purified antibodies; 5) Antibody-oligonucleotide coupling: The purified antibody obtained in step 4) is coupled with the oligonucleotide obtained in step 2) at a molar ratio of 1:5, and the reaction is carried out at 4° C. in the dark for 12 hours to finally obtain an antibody complex coupled with oligonucleotide.

[0037] 2. TdT-catalyzed ssDNA in situ synthesis 1) Prepare TdT enzyme, dNTP, chemically modified dNTP, dATP, and chemically modified dATP; 2) Using the unconjugated antibody oligonucleotide as a negative control, the oligonucleotide-conjugated antibody complex (2 ng / mL) obtained in step 1 of the TdT enzyme test was used to catalyze the synthesis of dNTP, chemically modified dNTP, dATP, and chemically modified dATP. The enzymatic catalytic system is shown in Table 1.

[0038]

[0039] 3) The prepared reaction liquid was reacted according to the following procedures: 30°C, 10 min; 98°C, 2 min. Finally, dNTP, chemically modified dNTP, dATP, and chemically modified dATP were used as substrates, and enzymatic synthesis was performed on 2 ng / mL oligonucleotide-coupled antibodies. The enzymatic ssDNA in situ synthesis product obtained was the antibody-coupled oligonucleotide AOC.

[0040] 3. Electrophoresis detection of enzymatic ssDNA in situ synthesis products 1) Add 30 μL TCEP to the product of enzymatic ssDNA in situ synthesis obtained in step 2 above, incubate at 40°C for 10 min and then at 98°C for 2 min; 2) After the reaction is completed, centrifuge at 12000 rpm for 10 min and transfer the supernatant to a concentrator tube; 3) Use a vacuum concentrator to concentrate until the tube wall is dry and free of liquid, and resuspend in 15 μL ddH2O; 4) Prepare 8% denaturing polyacrylamide gel and perform electrophoresis on the product of the previous step at 260V for 1h; Test results see Figure 2 Lane 1 is DNA Ladder, and lanes 2-5 are the products of enzymatic synthesis using dNTP, chemically modified dNTP, dATP, and chemically modified dATP as substrates on 2ng / mL oligonucleotide-coupled antibody. The gel image results show that ssDNA can be successfully enzymatically synthesized on the oligonucleotide-coupled antibody complex.

[0041] 4. Signal detection After the product synthesized using chemically modified dNTP or dATP as raw material in step 2 is bound to the antigen, the fluorescence intensity is detected using an electrochemiluminescence instrument. The results are shown in Table 2.

[0042] In Table 2, the product of enzymatic ssDNA in situ synthesis using antibodies as carriers can detect the expected fluorescence intensity, indicating that TdT enzymatic DNA in situ synthesis using antibodies as carriers can be achieved, and the modified oligonucleotides coupled to the antibodies can replace the secondary antibody to directly detect the target substrate.

[0043]

[0044] Note: * represents chemical modification.

[0045] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for in situ synthesis of ssDNA of antibody-coupled oligonucleotides, characterized in that: The following steps are involved: 1) Oligonucleotide synthesis: conventional chemical method to synthesize oligonucleotide chains; 2) Coupling of antibody and oligonucleotide: coupling the oligonucleotide in step 1) to the antibody via a covalent bond to form a complex; 3) TdT enzymatic in situ ssDNA synthesis: Using deoxyribonucleoside triphosphate monomers as raw materials, TdT enzyme is used to extend the 3'-OH end of the oligonucleotide of the complex obtained in step 2) to obtain the enzymatic in situ ssDNA synthesis product.

2. The method according to claim 1, characterized in that Step 3) The dNTPs used in the in situ synthesis of ssDNA are conventional dNTPs or chemically modified dNTPs.

3. The method according to claim 2, characterized in that The chemically modified group is located at the NH2 group on the A, G, C or T base.

4. The method according to claim 1 or 2, characterized in that: Step 3) The ssDNA in situ synthesis reaction system is: 6 μL antibody, final concentration is 2 ng / mL; 3 μL 10×Tris-HCl, final concentration is 0.1 M; 3 μL CoCl 2 ， Final concentration is 1mM; 3μL Triton, final concentration is 0.1%; 3μL dNTP, final concentration is 0.5mM; 3μL TdT, final concentration is 0.5mg / mL; ddH2O is added to 30μL; The reaction conditions for the ssDNA in situ synthesis are 30° C., 10 min; and 98° C., 2 min.

5. The in-situ synthesized product prepared by the method of claim 1.

6. Application of TdT enzyme in antibody-coupled oligonucleotide enzymatic ssDNA in situ synthesis.

7. Use of the method according to claim 1 in antibody-coupled oligonucleotide enzymatic ssDNA in situ synthesis.

8. Use of the method according to claim 1 in enhancing antibody recognition ability.

9. Use of the in situ synthesized product according to claim 5 in enhancing antibody recognition ability.

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