CN-121992051-A - Method for preparing long-chain double-stranded nucleic acid

Abstract

The present application relates to a method for synthesizing long-chain double-stranded nucleic acid from short-chain single-stranded nucleic acid, which is characterized by the presence of an intermediate, which is referred to as long-chain double-stranded nucleic acid with a split. In addition, the application relates to the use of said method for synthesizing long-chain double-stranded nucleic acids.

Inventors

• Xia Ninuo
• WEI DIMING
• WU ZHIGUANG
• WANG YAQI

Assignees

• 合肥核信生物科技有限责任公司
• 清华大学

Dates

Publication Date

20260508

Application Date

20241108

Claims (17)

1. 1. A method of synthesizing long-chain double-stranded nucleic acid from short-chain single-stranded nucleic acid, the method comprising the steps of: a) The short chain single chain nucleic acid is complementarily paired to form an intermediate, and the intermediate is the long chain with the split Double-stranded nucleic acid, wherein the breach refers to the absence of phosphodiester between two adjacent nucleic acids in the long-chain double-stranded nucleic acid Ester bonds; b) Repairing the split in the intermediate to obtain the long-chain double-stranded nucleic acid.
2. 2. The method of claim 1, wherein the short-chain single-stranded nucleic acid comprises 10-300 nucleotides.
3. 3. The method according to claim 1, wherein the length of the long-chain double-stranded nucleic acid is more than 2 times, preferably more than 5 times, more preferably more than 10 times the length of the short-chain single-stranded nucleic acid.
4. 4. The method of claim 1, wherein the short-chain single-stranded nucleic acid is designed and synthesized by: (1) Designing a nucleotide sequence comprising the long-chain double-stranded nucleic acid; (2) Dividing each single strand of the double strands of the nucleotide sequence into a plurality of short-strand single-strand nuclei Acid, and (3) Synthesizing the short-chain single-stranded nucleic acid.
5. 5. The method of claim 4, wherein in step (2), the dividing point of the two strands is offset from any dividing point of the other strand by at least 10 nucleotides.
6. 6. The method of claim 1, wherein in step b) the repairing is by forming a phosphodiester bond at the split.
7. 7. The method of claim 6, wherein the intermediate is a linear nucleic acid, and the method of repairing comprises: i. Attaching the intermediate to a support; transferring said vector into a host.
8. 8. The method of claim 6, wherein the intermediate is a circular nucleic acid.
9. 9. The method of claim 8, wherein the circular nucleic acid is a plasmid and the method of repairing comprises transferring the plasmid into a host.
10. 10. The method of claim 1, the number of breaks being 2-50.
11. 11. The method of claim 1, the number of breaks being 51 and above 51.
12. 12. The method of claim 11, further comprising, prior to step i, treating the intermediate with a ligase.
13. 13. Use of the method of any one of claims 1-12 in the synthesis of long-chain double-stranded nucleic acids.
14. 14. The use of claim 13, wherein the number of long-chain double-stranded nucleic acids is 1.
15. 15. The use according to claim 13, wherein the number of long-chain double stranded nucleic acids is between 2 and 1000, preferably between 2 and 500, more preferably between 2 and 100.
16. 16. The use of claim 13, wherein the long-chain double-stranded nucleic acid is a linear nucleic acid.
17. 17. The use of claim 13, wherein the long-chain double-stranded nucleic acid is a circular nucleic acid.

Description

Method for preparing long-chain double-stranded nucleic acid Technical Field The application relates to the field of biological medicine, in particular to an improved method for synthesizing nucleic acid from head, and more particularly relates to a method for synthesizing long-chain double-chain nucleic acid from short-chain single-chain nucleic acid. Background Nucleic acids are synthesized from the head, i.e.from the basic nucleotide units, to construct entirely new DNA or RNA molecules, which is of great importance for synthetic biology, gene therapy and biotechnology. It not only provides a powerful tool for researching gene functions and regulatory mechanisms, but also enables scientists to design and construct synthetic genes and regulatory elements with specific functions, promoting understanding of life origins and evolution. In the medical field, nucleic acid de novo synthesis is useful for the development of personalized medicine and vaccines, providing a new strategy for the treatment of genetic diseases and cancers. In addition, the technology improves crops by synthesizing genes with specific characters in agricultural biotechnology, and enhances the resistance and yield of the crops. De novo synthesis of nucleic acids also provides an accurate means of genetic analysis for forensics, helping to solve the problem of genetic evidence in criminal investigation. Along with the reduction of synthesis cost and the improvement of synthesis efficiency, the nucleic acid first synthesis technology is becoming an important support for the research and application of bioscience, and the cognitive boundary of people on life science is continuously expanded. There are different solutions for short-chain nucleic acids and long-chain nucleic acids with respect to specific de novo synthesis methods. For short-chain nucleic acids, chemical methods using automated nucleic acid synthesizers are mainly used. For long-chain nucleic acids, this is currently achieved mainly by a method of synthesizing short-chain nucleic acids first and then assembling them into long-chain nucleic acids. The assembly methods currently used are mainly carried out by enzymes, such as polymerase cycle assembly (polymerase cycle assembly, PCA) and ligase-based assembly (ligase based assembly, LBA), however, the current synthesis methods have problems such as nucleic acid sequence mismatches, which can be partially solved by the introduction of high fidelity enzymes. We still need to explore more suitable schemes. Disclosure of Invention The present application provides a novel method for synthesizing long-chain double-stranded nucleic acid from short-chain single-stranded nucleic acid. As shown in FIG. 1, the application adopts the synthesis path of short-chain single-chain nucleic acid-split intermediate-long-chain double-chain nucleic acid, so that the short-chain single-chain nucleic acid (10-300 bp) can be assembled into long-chain double-chain nucleic acid (more than 500 bp) effectively with low mismatch rate, and the yield of the long-chain double-chain nucleic acid is further increased by means of nucleic acid amplification and the like. In the current synthesis of long fragments of long-chain double-stranded nucleic acid from short-chain single-stranded nucleic acid mainly comprising PCA and LBA, the annealing process of the short-chain single-stranded nucleic acid and the synthesis of phosphodiester bond are performed in parallel, namely, the short-chain single-stranded nucleic acid is subjected to polymerization extension (for PCA) or split connection (for LBA) of nucleic acid when the reverse complementary pairing region is formed locally, in which case, once mismatch is caused by high homology of each fragment of short-chain single-stranded nucleic acid (namely, homology of less than 100% of the reverse complementary region), the mismatch is fixed on the product, and accuracy of long-chain double-stranded nucleic acid sequence is reduced, and for short-chain single-stranded nucleic acid comprising special sequence (self-pairing, high GC or repeated sequence), not only mismatch is likely to occur, but also some stem-loop structures as shown in FIG. 5B and FIG. 8A are likely to be formed, and once the stem-loop structures are fixed on the product are likely to reduce accuracy of the final product, and assembly failure results are likely to occur. Unlike the prior art, the method of the present application separates the annealing process from the process of synthesizing phosphodiester bonds, i.e., the short-chain single-stranded nucleic acid is annealed to form a split-carrying intermediate, and then the split is stitched to form the complete long-chain double-stranded nucleic acid. Wherein the intermediate gives some buffer space for the reverse complement region of the mismatch, and the mismatch is not directly immobilized, and the short-chain single-stranded nucleic acid is not changed after the intermediate is formed, but i