CN115605590A - Method for preparing site-directed modified long-chain DNA - Google Patents

Abstract

Translated fromChinese

提供了一种制备长链DNA的方法，包括：合成步骤、退火步骤，和连接步骤。所述方法通过对第一链和第二链中DNA片段的设计，能够实现对长链DNA中任意精确位点的化学修饰，使长链DNA获得提高的稳定性和改善的免疫原性等性能。通过该方法能够得到由连续化的单链DNA与片段化的单链DNA互补形成的双链DNA的组装体，双链DNA的组装体仅需通过简单变性即可得到单链的长链DNA，有效简化了目前长链DNA的合成步骤，提高了合成效率，适合于工业化大规模制备。

Provided is a method for preparing long-chain DNA, comprising: a synthesis step, an annealing step, and a linking step. The method can realize the chemical modification of any precise site in the long-chain DNA through the design of the DNA fragments in the first strand and the second strand, so that the long-chain DNA can obtain properties such as improved stability and improved immunogenicity . Through this method, a double-stranded DNA assembly formed by the complementarity of continuous single-stranded DNA and fragmented single-stranded DNA can be obtained. The double-stranded DNA assembly can be obtained by simple denaturation. Single-stranded long-chain DNA, The method effectively simplifies the synthesis steps of the current long-chain DNA, improves the synthesis efficiency, and is suitable for large-scale industrial preparation.

Description

Translated fromChinese

PCT国内申请，说明书已公开。PCT domestic application, specification has been published.

Claims (16)

1. A method for preparing long-chain DNA, which comprises the following steps:

   the synthesis steps are as follows: synthesizing a first-strand DNA fragment group and a second-strand DNA fragment group, wherein the first-strand DNA fragment group comprises a DNA fragment n_i And DNA fragment n_i+1 The DNA fragment group of the second strand comprises a DNA fragment m_i (ii) a i is selected from positive integers of more than 1; wherein, the DNA fragment m_i 5' terminal sequence of (1) and DNA fragment n_i+1 The 5' terminal sequence of (a) is a complementary sequence, a DNA fragment m_i The 3' terminal sequence of (A) and the DNA fragment n_i The 3' terminal sequence of (a) is a complementary sequence;

   and (3) annealing: mixing the DNA fragment group of the first chain and the DNA fragment group of the second chain in the same reaction system, and annealing to form an assembly precursor of double-stranded DNA; wherein a connecting port exists between two adjacent DNA fragments in the first strand, and a connecting port exists between two adjacent DNA fragments in the second strand; the connecting ports between the adjacent DNA fragments in the first strand DNA fragment group and the connecting ports between the adjacent DNA fragments in the second strand DNA fragment group are staggered;

   a connection step: connecting the connecting ports of the single-stranded DNAs of the first strand and the second strand to obtain an assembly of double-stranded DNAs formed by complementing the continuous single-stranded DNAs and the fragmented single-stranded DNAs.
2. The method for preparing long-chain DNA according to claim 1, further comprising the steps of:

   a denaturation step: performing denaturation treatment on the assembly of the double-stranded DNA to obtain continuous single-stranded DNA;

   optionally, the method further comprises a purification step: purifying the continuous single-stranded DNA from the reaction system.
3. The method for preparing long-chain DNA according to claim 1 or 2,

   the DNA fragment n_i+1 3' terminal sequence of (1) andthe 3' terminal sequence of the other DNA fragment of the second strand is a complementary sequence;

   alternatively, the DNA fragment n_i+1 The 3' terminal sequence of (3) and the DNA fragment m_i+1 The 3' terminal sequence of (a) is a complementary sequence, the DNA fragment m_i+1 Is complementary to other DNA fragments of the first strand DNA fragment set or is an unpaired sequence.
4. The method for preparing long-chain DNA according to any one of claims 1 to 3, wherein the length of the continuous single-stranded DNA is 60nt or more, preferably 60 to 1000nt.
5. The method for preparing long-chain DNA according to any one of claims 1 to 4, wherein the length of any one of the DNA fragments in the group of DNA fragments of the first strand and the group of DNA fragments of the second strand is 8 to 120nt, preferably 10 to 80nt, more preferably 15 to 40nt, and most preferably 20 to 30nt.
6. The method for producing a long-chain DNA according to any one of claims 1 to 5, wherein the length of the 5' -end sequence of any one of the DNA fragments in the group of DNA fragments of the first strand and the group of DNA fragments of the second strand is 4nt or more, preferably 4 to 50nt, more preferably 6 to 30nt, most preferably 10 to 20nt; or,

   the length of the 3' end sequence of any one DNA fragment in the first strand DNA fragment group and the second strand DNA fragment group is more than 4nt, preferably 4-50nt, more preferably 6-30nt, and most preferably 10-20nt.
7. The method for preparing a long-chain DNA according to any one of claims 1 to 6, wherein any one of the DNA fragments in the group of DNA fragments of the first chain comprises a phosphate group at the 5 'terminal, and a hydroxyl group at the 3' terminal; in the connecting step, phosphate groups and hydroxyl groups on two sides of the connecting port are connected to form phosphodiester bonds;

   optionally, adjacent phosphate groups and hydroxyl groups in the first strand are joined as phosphodiester linkages with enzymatic or chemical ligation.
8. The method for preparing a long-chain DNA according to any one of claims 1 to 6, wherein any one of the DNA fragments in the group of DNA fragments of the second chain comprises a phosphate group at the 5 'terminal, and a hydroxyl group at the 3' terminal; in the connecting step, phosphate groups and hydroxyl groups on two sides of the connecting port are connected to form phosphodiester bonds;

   optionally, adjacent phosphate groups and hydroxyl groups in the second strand are joined as phosphodiester linkages by enzymatic or chemical ligation.
9. The method for preparing a long-chain DNA according to any one of claims 1 to 8, wherein one or more positions of any one of the DNA fragments in the group of DNA fragments of the first strand and the group of DNA fragments of the second strand comprise a modified base, and the base at a position adjacent to the junction port is an unmodified base;

   alternatively, the modification is selected from m⁶ A、Ψ、m¹ A、m⁵ A、ms² i⁶ A、i⁶ A、m³ C、m⁵ C、ac⁴ C、m⁷ G、 m2,2G、m² G、m¹ G、Q、m⁵ U、mcm⁵ U、ncm⁵ U、ncm⁵ Um、D、mcm⁵ s² U、Inosine(I)、hm⁵ C、s⁴ U、s² U, azobenzene, cm, um, gm, t⁶ A、yW、ms² t⁶ A or a derivative thereof.
10. The method for preparing a long-chain DNA according to any one of claims 1 to 9, wherein the modified deoxyribose is contained at one or more positions of any one of the group of DNA fragments of the first strand and the group of DNA fragments of the second strand, and the deoxyribose at a position adjacent to the connection port is an unmodified deoxyribose;

    alternatively, the modification is selected from LNA, 2' -OMe, 3' -OMe u, vmoe, 2' -F or 2' -OBn (2 ' -O-benzyl group) or a derivative thereof.
11. The method for producing a long-chain DNA according to any one of claims 1 to 10, wherein one or more positions of any one of the group of DNA fragments of the first strand and the group of DNA fragments of the second strand contain a modified phosphodiester bond, and the phosphodiester bond at a position adjacent to the connection port is an unmodified phosphodiester bond;

    optionally, the modification is selected from Phosphothioate (PS).
12. The method for preparing a long-chain DNA according to any one of claims 1 to 11, wherein in the annealing step, the temperature is reduced after the incubation of the group of DNA fragments of the first chain and the group of DNA fragments of the second chain, so as to form an assembly precursor of a double-stranded DNA;

    optionally, the temperature of the incubation is any temperature from 0 to 100 ℃, preferably any temperature from 10 to 85 ℃, more preferably any temperature from 20 to 65 ℃.
13. The method for producing a long-chain DNA according to any one of claims 1 to 12, wherein in the annealing step, the reaction system is obtained by dissolving the group of DNA fragments of the first strand and the group of DNA fragments of the second strand in the same solvent.
14. The method for producing long-chain DNA according to claim 13, wherein the reaction system has a pH of 3 to 11, preferably a pH of 4 to 10, more preferably a pH of 5 to 9, and most preferably a pH of 6 to 8.
15. The method for preparing long-chain DNA according to claim 13 or 14, wherein the molar ratio of any two DNA fragments in the group of DNA fragments of the first strand and the group of DNA fragments of the second strand in the reaction system is 1: (0.1-10), preferably 1: (0.5-1), most preferably 1:1.
16. a long-chain DNA prepared by the method according to any one of claims 1 to 15, wherein the long-chain DNA is a single-stranded long-chain DNA;

    preferably, the long-chain DNA comprises a modified base, ribose, or phosphodiester linkage at one or more positions.

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