WO2022170707A1 - Procédé de préparation d'adn à chaîne longue modifié dirigé sur site - Google Patents

Procédé de préparation d'adn à chaîne longue modifié dirigé sur site Download PDF

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WO2022170707A1
WO2022170707A1 PCT/CN2021/098430 CN2021098430W WO2022170707A1 WO 2022170707 A1 WO2022170707 A1 WO 2022170707A1 CN 2021098430 W CN2021098430 W CN 2021098430W WO 2022170707 A1 WO2022170707 A1 WO 2022170707A1
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dna
strand
dna fragment
group
chain
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Chinese (zh)
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刘冬生
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • the present disclosure belongs to the fields of molecular biology and synthetic biology, and in particular, the present disclosure relates to a method for preparing long-chain DNA and the prepared long-chain DNA.
  • DNA molecules play a vital role in the field of life sciences.
  • the vast majority of biological research and bioengineering require the participation of DNA molecules of different lengths, including oligonucleotides and longer constructs, such as synthetic genes, chromosomes, etc. [1,2] .
  • DNA molecules can also be used in supramolecular polymerization [3] , nanotechnology [4] and information storage [5] and other fields. Therefore, the cost-effective synthesis of DNA molecules is an important issue in the field of life sciences.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • the ligase-based method can splicing short-chain DNA into long-chain DNA by step-by-step splicing [13,14] , This kind of method needs to be carried out in multiple steps, and needs to be purified before each splicing, which is complicated, expensive and small in yield;
  • another method based on ligase is ligase chain reaction (LCR) [15] , It relies on a relatively expensive heat-resistant DNA ligase, and the product drag is more serious, and the target DNA double-strand needs to be amplified by PCR; in addition, polymerase-based assembly methods include overlap extension polymerase chain reaction (OE-PCR).
  • PCA polymerase stepwise assembly
  • CPEC circular polymerase extension method
  • the second is to introduce a primer whose 5' end has been modified with phosphoric acid in PCR amplification, and then use Lambda exonuclease to selectively digest a 5' phosphoric acid-modified single-stranded DNA in the double-stranded DNA. , and obtain the complementary single-stranded DNA without phosphate modification [23] , but this method has the disadvantage of incomplete digestion of single-stranded DNA, and it will become more serious with the increase of DNA chain length; third, through denaturing polypropylene Amide gel electrophoresis (PAGE) is used for gel cutting purification.
  • PAGE denaturing polypropylene Amide gel electrophoresis
  • one primer is modified during double-stranded amplification, such as the introduction of cleavable ribose residues and pH-unstable clips, etc.
  • the strands run at different speeds during electrophoresis, allowing for gel excision; however, this approach makes denaturation of the duplex more difficult as the length of the DNA strand increases, and the resolution of PAGE for both strands decreases. It is difficult to obtain high-purity DNA single-stranded.
  • all of the methods described above are difficult to introduce modifications at specific positions on either the DNA single strand or the DNA double strand.
  • DNA modifications generally including modifications to bases, phosphodiester bonds and deoxyribose [24-29] ; at present, for DNA modified at specific sites, it is generally only possible to introduce modified monomers through chemical synthesis. It is suitable for modifying short-chain DNA. Therefore, how to efficiently synthesize single-stranded long-chain DNA and further perform precise modification of long-chain DNA at any site is a technical problem that needs to be solved urgently in the art.
  • the method for synthesizing long-chain DNA based on solid-phase synthesis has low yield, high cost, and high error rate when the chain length is long, and the synthesis of long-chain DNA based on DNA polymerase relies on The problem of high-fidelity polymerase and the inability to achieve precise modification of specific bases.
  • the present disclosure provides a method for preparing long-chain DNA, which is independent of DNA polymerase and capable of synthesizing long-chain DNA with arbitrary sequences.
  • the method provided by the present disclosure is suitable for precise modification of specific sites of long-chain DNA, and has the advantages of low synthesis difficulty, high accuracy and low cost.
  • the method provided by the present disclosure can obtain single-stranded long-chain DNA modified at any site, and the long-chain DNA has high synthesis efficiency and high purity, and is suitable for 60 nt or more, especially in the range of 60-1000 nt. synthesis of single-stranded DNA.
  • the present disclosure provides a method for preparing long-chain DNA, including the following steps:
  • Synthesis step synthesizing the first-strand DNA fragment group and the second-strand DNA fragment group, the first-strand DNA fragment group including DNA fragment n i and DNA fragment n i+1 , the second-strand DNA fragment group
  • the group includes DNA fragment m i ; i is selected from a positive integer of 1 or more; wherein, the 5'-end sequence of the DNA fragment mi and the 5'-end sequence of the DNA fragment n i +1 are complementary sequences, and the 3' end sequence of the DNA fragment mi The end sequence and the 3' end sequence of the DNA fragment n i are complementary sequences;
  • Annealing step mixing the DNA fragment group of the first strand and the DNA fragment group of the second strand in the same reaction system, and annealing to form an assembly precursor of double-stranded DNA; wherein, the first strands are adjacent to each other. There is a connection port between the two DNA fragments of The junctions with the adjacent DNA fragments in the DNA fragment group of the second strand are staggered from each other;
  • Connecting step connecting the connection port of any single-stranded DNA in the first strand and the second strand to obtain a double-stranded DNA assembly formed by the complementation of the continuous single-stranded DNA and the fragmented single-stranded DNA.
  • the method for preparing long-chain DNA according to the present disclosure further comprises the following steps:
  • Denaturation step denaturation of the double-stranded DNA assembly to obtain continuous single-stranded DNA
  • the method further includes a purification step: purifying the continuous single-stranded DNA from the reaction system.
  • the 3'-end sequence of the DNA fragment n i+1 and the 3'-end sequence of the other DNA fragments of the second strand are complementary sequence;
  • sequence at the 3' end of the DNA fragment n i+1 and the sequence at the 3' end of the DNA fragment m i+1 are complementary sequences, and the sequence at the 5' end of the DNA fragment m i+1 is the same as the sequence at the 3' end of the DNA fragment m i+1.
  • the other DNA fragments of the set of DNA fragments of one strand are either complementary sequences or unpaired sequences.
  • the length of the continuous single-stranded DNA is 60nt or more, preferably 80nt or more, preferably 100nt or more, more preferably 60-1000nt, more Preferably 80-600nt, more preferably 100-400nt, most preferably 120-360nt.
  • the method for preparing long-chain DNA according to the present disclosure wherein the length of any DNA fragment in the first-strand DNA fragment group and the second-strand DNA fragment group is 8- 120nt, preferably 10-80nt, more preferably 15-40nt, most preferably 20-30nt.
  • the method for preparing long-chain DNA according to the present disclosure wherein the 5'-end sequence of any DNA fragment in the first-strand DNA fragment group and the second-strand DNA fragment group
  • the length is 4nt or more, preferably 4-50nt, more preferably 6-30nt, most preferably 10-20nt; or,
  • the length of the 3'-end sequence of any DNA fragment in the DNA fragment group of the first strand and the DNA fragment group of the second strand is 4 nt or more, preferably 4-50 nt, more preferably 6-30 nt, most preferably 10- 20nt.
  • the method for preparing long-chain DNA wherein any DNA fragment in the set of DNA fragments of the first strand comprises a phosphate group at the 5' end, and a phosphate group at the 3' end
  • the phosphoric acid groups and hydroxyl groups on both sides of the connecting port are connected as phosphodiester bonds;
  • adjacent phosphate groups and hydroxyl groups in the first strand are linked as phosphodiester bonds by enzymatic or chemical linkage.
  • the method for preparing long-chain DNA according to the present disclosure wherein any DNA fragment in the group of DNA fragments of the second strand comprises a phosphate group at the 5' end, and a phosphate group at the 3' end
  • the phosphoric acid groups and hydroxyl groups on both sides of the connecting port are connected as phosphodiester bonds;
  • adjacent phosphate groups and hydroxyl groups in the second strand are linked as phosphodiester bonds by enzymatic or chemical linkage.
  • the method for preparing long-chain DNA according to the present disclosure wherein one or more of any DNA fragments in the first-strand DNA fragment group and the second-strand DNA fragment group comprising a modified base at the position, and the base at the position immediately adjacent to the junction is an unmodified base;
  • the modification is selected from m 6 A, ⁇ , m 1 A, m 5 A, ms 2 i 6 A, i 6 A, m 3 C, m 5 C, ac 4 C, m 7 G, m 2 ,2G, m2G , m1G , Q, m5U , mcm5U , ncm5U , ncm5Um , D, mcm5s2U , Inosine (I), hm5C , s4U , s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or derivatives thereof.
  • the method for preparing long-chain DNA according to the present disclosure wherein one or more of any DNA fragments in the first-strand DNA fragment group and the second-strand DNA fragment group comprising modified deoxyribose at the position, and the deoxyribose at the position immediately adjacent to the junction is unmodified deoxyribose;
  • the modification is selected from LNA, 2'-OMe, 3'-OMeU, vmoe, 2'-F or 2'-OBn (2'-O-benzyl group) or derivatives thereof.
  • the method for preparing long-chain DNA according to the present disclosure wherein one or more of any DNA fragments in the first-strand DNA fragment group and the second-strand DNA fragment group comprising a modified phosphodiester bond at the position, and the phosphodiester bond at the position immediately adjacent to the connecting port is an unmodified phosphodiester bond;
  • the modification is selected from phosphorothioate (PS).
  • the incubation temperature is any temperature of 0-100°C, preferably any temperature of 10-85°C, more preferably any temperature of 20-65°C, and the incubation time is any desired time;
  • the speed of the cooling can be any speed, and the temperature can be any temperature at which the DNA fragments in the reaction system are hybridized to form an assembly precursor of double-stranded DNA.
  • the method for preparing long-chain DNA according to the present disclosure wherein, in the annealing step, the first-strand DNA fragment group and the second-strand DNA fragment group are dissolved in the same solvent to obtain the reaction system.
  • the pH of the reaction system is 3-11, preferably pH 4-10, more preferably pH 5-9, most preferably pH 6- 8.
  • any two DNA fragments in the first-strand DNA fragment group and the second-strand DNA fragment group is 1:(0.1-10), preferably 1:(0.5-1), most preferably 1:1.
  • the present disclosure also provides a long-chain DNA, wherein the long-chain DNA is prepared by the method according to the present disclosure, and the long-chain DNA is a single-stranded long-chain DNA;
  • the long strand of DNA comprises modified base, ribose or phosphodiester linkages at one or more positions.
  • the method for preparing long-chain DNA can prepare long-chain DNA of any sequence, and realize precise modification of any site in the long-chain DNA, and the preparation method of long-chain DNA uses raw materials DNA is a template, does not rely on exogenous DNA, and does not rely on DNA polymerase, etc., has the advantages of low cost, low synthesis difficulty, high yield, and high sequence accuracy, and is suitable for large-scale popularization.
  • the present disclosure provides a method for preparing long-stranded DNA by preparing an assembly of double-stranded DNA formed by complementation of continuous single-stranded DNA and fragmented single-stranded DNA, and the assembly of double-stranded DNA is only
  • the target long-chain DNA can be obtained by simple denaturation treatment.
  • the denaturation difficulty of the double-stranded DNA assembly in the present disclosure is low; and, for non-targeted DNA
  • the single-stranded DNA is dispersed in the reaction system as DNA fragments after the preparation is completed, and it does not need to be sheared or reprocessed, which effectively simplifies the preparation steps of single-stranded long-chain DNA, and improves the preparation efficiency and efficiency. Synthetic purity of single-stranded long-chain DNA.
  • the method for preparing long-chain DNA provided by the present disclosure, in which precise insertion of modified bases at any site is achieved, solves the problem that the current long-chain DNA synthesis method cannot achieve precise modification of specific sites. question.
  • the long-chain DNA provided by the present disclosure is a single-stranded long-chain DNA prepared by the above-mentioned preparation method, and its sequence accuracy is high, and can be modified at any site to obtain improved stability, immunity and immunity. It has a wide range of application prospects in drug research and development, clinical treatment, etc.
  • Figure 1 shows a schematic diagram of the process of preparing long-chain DNA
  • Figure 2 shows a schematic diagram of the assembly of DNA 100/80bp
  • Figure 3 shows the results of gradient native polyacrylamide gel electrophoresis characterization results of DNA 100/80bp assemblies
  • Figure 4 shows the characterization results of denaturing polyacrylamide gel electrophoresis of 100nt DNA single strands
  • Figure 5 shows the comparison of the characterization results of native and denaturing polyacrylamide gel electrophoresis of DNA 100/80bp assemblies without ligation
  • Figure 6 shows the characterization results of non-denaturing polyacrylamide gel electrophoresis and fragment analyzer characterization results of length-extended double-stranded DNA assemblies.
  • numerical range represented by "numerical value A to numerical value B" refers to the range including the numerical values A and B at the endpoints.
  • multiple in “multiple”, “plurality”, “plurality”, etc. means a numerical value of 2 or more.
  • the "substantially”, “substantially” or “substantially” means that the error is less than 5%, or less than 3% or less than 1% compared to the relevant perfect standard or theoretical standard.
  • water includes tap water, deionized water, distilled water, double-distilled water, purified water, ion-exchanged water, and the like, any practicable water.
  • double-stranded DNA assembly and “double-stranded DNA” have the same meaning and can be substituted for each other.
  • connection port is also called a nick, which exists between two adjacent deoxyribonucleotides in single-stranded DNA because the gap between the two adjacent deoxyribonucleotides is Produced without the formation of phosphodiester bonds.
  • a first aspect of the present disclosure provides a method for preparing long-chain DNA, as shown in FIG. 1 , which includes the following steps:
  • Synthesis step synthesizing the first-strand DNA fragment group and the second-strand DNA fragment group, the first-strand DNA fragment group including DNA fragment n i and DNA fragment n i+1 , the second-strand DNA fragment group
  • the group includes DNA fragment m i ; i is selected from a positive integer of 1 or more; wherein, the 5'-end sequence of the DNA fragment mi and the 5'-end sequence of the DNA fragment n i +1 are complementary sequences, and the 3' end sequence of the DNA fragment mi The end sequence and the 3' end sequence of the DNA fragment n i are complementary sequences;
  • Annealing step mixing the DNA fragment group of the first strand and the DNA fragment group of the second strand in the same reaction system, and annealing to form an assembly precursor of double-stranded DNA; wherein, the first strands are adjacent to each other. There is a connection port between the two DNA fragments of The junctions with the adjacent DNA fragments in the DNA fragment group of the second strand are staggered from each other;
  • Connecting step connecting the connection port of any single-stranded DNA in the first strand and the second strand to obtain a double-stranded DNA assembly formed by the complementation of the continuous single-stranded DNA and the fragmented single-stranded DNA.
  • DNA is often synthesized by solid-phase synthesis.
  • synthesis of long double-stranded DNA by means of short fragment annealing, nick ligation and PCR amplification, but its reliance on high-fidelity DNA polymerases easily leads to increased costs and increased error rates.
  • Patent document CN102876658A invented a method for large-scale synthesis of long-chain nucleic acid molecules, and its specific steps include: the first step, the synthesis of short-chain nucleic acid molecule fragments; the second step, the chemical modification of the endpoint; the third step, the short chain The connection of nucleic acid molecule fragments; the fourth step, the purification of DNA double strands; the fifth step, the use of nucleic acid amplification technology to amplify and amplify the target long-chain DNA molecule.
  • the above method can realize the synthesis of long-chain DNA, it relies on PCR for amplification and amplification, and the prepared long-chain DNA is a double-stranded DNA formed by the complementation of two continuous single-stranded DNAs.
  • the long-chain DNA cannot be modified at the entry site; It is difficult to denature double-stranded DNA formed by long chains, and it is difficult to obtain single-stranded long-chain DNA.
  • a reaction system in which two continuous single-stranded DNAs are mixed is obtained, which affects the recovery efficiency and recovery purity of the target single-stranded DNA.
  • there may be unsuccessful ligation of multiple junction ports in the purified assembly and only a small part of complete double-stranded DNA is formed, so the yield of double-stranded DNA is low.
  • the preparation method of the present disclosure divides the long-chain DNA into several short DNA fragments, which greatly reduces the difficulty of synthesizing the long-chain DNA.
  • DNA polymerase which effectively reduces the difficulty of chemical synthesis of long single-stranded DNA, and can realize the synthesis of base, deoxyribose or phosphodiester at any position in the long single-stranded DNA.
  • the modification of the bond has the advantages of low cost, high yield and high sequence accuracy.
  • the preparation method of the present disclosure only connects the junction of one DNA strand in the first strand and the second strand to obtain a double-stranded DNA assembly formed by the complementarity of the continuous single-stranded DNA and the fragmented single-stranded DNA .
  • the preparation method also does not include an amplification step, and avoids connecting the junction of the fragmented single-stranded DNA in the double-stranded DNA assembly, thereby realizing the preparation of long-chain DNA independent of the PCR amplification step, and can obtain higher yield.
  • the assembly of double-stranded DNA can recover the target long-stranded DNA only by simple denaturation treatment.
  • the preparation method does not include digestion and shearing of non-target single-stranded DNA, which effectively improves the single-stranded DNA.
  • the preparation efficiency and purity of long-chain DNA are suitable for large-scale industrial applications.
  • FIG. 2 shows a double-stranded long-chain DNA structure.
  • the double-stranded DNA is composed of at least partially complementary first and second strands, wherein the first or second strand is a target synthesized long-chain DNA.
  • the nucleotide sequences of the first strand and the second strand are respectively divided, so that the nucleotide sequences of the first strand and the second strand are divided into DNA fragment sequences of several short strands.
  • the DNA segment group of the first strand includes DNA segment n i and DNA segment n i+1
  • the DNA segment group of the second strand includes DNA segment mi .
  • the 5'-end sequence of the DNA fragment mi and the 5'-end sequence of the DNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the DNA fragment mi and the 3'-end sequence of the DNA fragment n i are complementary sequences.
  • the 5'-end sequence of DNA fragment n i and the 5'-end sequence of other DNA fragments of the second strand are complementary sequences or unpaired sequences
  • the 3'-end sequence of DNA fragment n i+1 is the same as that of other DNA fragments of the second strand.
  • the 3'-end sequence is either a complementary sequence or an unpaired sequence.
  • the DNA segment group of the first strand may also include other DNA segments.
  • the DNA segment group of the first strand includes at least x DNA segments, where x is a positive integer greater than or equal to 2.
  • x is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Not exhaustive.
  • the set of DNA fragments of the first strand includes DNA fragment n i , DNA fragment n i+1 , DNA fragment n i+2 . In some embodiments, the set of DNA fragments of the first strand includes DNA fragment ni , DNA fragment ni+1 , DNA fragment ni+2 , DNA fragment ni+3 . In some embodiments, the set of DNA fragments of the first strand includes DNA fragment ni , DNA fragment ni+1 , DNA fragment ni+2 , DNA fragment ni+3 , DNA fragment ni+4 .
  • the DNA fragment group of the first strand may also include other numbers of DNA fragments, which are not exhaustive in the present disclosure.
  • the DNA segment group of the second strand may also include other DNA segments.
  • the DNA segment group of the first strand includes at least y DNA segments, and y is a positive integer greater than 1.
  • y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. This will not be exhaustive.
  • the set of DNA fragments of the second strand includes DNA fragments mi , DNA fragments mi+1 .
  • the 5'-end sequence of the DNA fragment mi and the 5'-end sequence of the DNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the DNA fragment mi and the 3'-end sequence of the DNA fragment n i are complementary sequences
  • the sequence at the 3' end of the DNA fragment m i +1 is complementary to the sequence at the 3' end of the DNA fragment n i+1
  • the sequence at the 5' end of the DNA fragment m i+1 is the same as the 5' end of the DNA fragment n i+2 .
  • the sequences are complementary or unpaired.
  • the set of DNA fragments of the second strand includes DNA fragment mi , DNA fragment mi+1 , DNA fragment mi+2 .
  • the 5'-end sequence of the DNA fragment mi and the 5'-end sequence of the DNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the DNA fragment mi and the 3'-end sequence of the DNA fragment n i are complementary sequences;
  • the sequence at the 3' end of the DNA fragment m i +1 is complementary to the sequence at the 3' end of the DNA fragment n i+1
  • the sequence at the 5' end of the DNA fragment m i+1 is the same as the 5' end of the DNA fragment n i+2 .
  • the sequence is a complementary sequence; the sequence at the 3' end of the DNA fragment mi+2 is a complementary sequence with the sequence at the 3' end of the DNA fragment n i+2 , and the sequence at the 5' end of the DNA fragment mi+2 is the same as the sequence at the 5' end of the DNA fragment n i+
  • the 5' sequence of 3 is either a complementary sequence or an unpaired sequence.
  • the set of DNA fragments of the second strand includes DNA fragment mi , DNA fragment mi+1 , DNA fragment mi+2 , DNA fragment mi+3 .
  • the 5'-end sequence of the DNA fragment mi and the 5'-end sequence of the DNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the DNA fragment mi and the 3'-end sequence of the DNA fragment n i are complementary sequences;
  • the sequence at the 3' end of the DNA fragment m i +1 is complementary to the sequence at the 3' end of the DNA fragment n i+1
  • the sequence at the 5' end of the DNA fragment m i+1 is the same as the 5' end of the DNA fragment n i+2 .
  • the sequence is a complementary sequence; the sequence at the 3' end of the DNA fragment mi+2 is a complementary sequence with the sequence at the 3' end of the DNA fragment n i+2 , and the sequence at the 5' end of the DNA fragment mi+2 is the same as the sequence at the 5' end of the DNA fragment n i+
  • the 5'-end sequence of 3 is a complementary sequence; the 3'-end sequence of the DNA fragment mi+3 is a complementary sequence with the 3'-end sequence of the DNA fragment n i +3 , and the 5'-end sequence of the DNA fragment mi+3 is the same as the
  • the 5'-end sequence of the DNA fragment n i+4 is a complementary sequence or an unpaired sequence.
  • the DNA fragment group of the second strand may also include other numbers of DNA fragments, which are not exhaustive in the present disclosure.
  • the 5'-end sequence and the 3'-end sequence refer to dividing the nucleotide fragment in the 5' to 3' direction, so that the nucleotide fragment is divided into two regions.
  • a sequence in a region near the 5' end is referred to as a 5' end sequence
  • a sequence in another region near the 3' end is referred to as a 3' end sequence.
  • the 5' terminus is a nucleotide in the 5' to 3' direction at the 5' endmost position in the nucleotide chain, which typically has a 5' terminus phosphate group.
  • the 3' terminus is a nucleotide in the 5' to 3' direction at the 3' endmost position in the nucleotide chain, which typically has a 3' terminus hydroxyl group.
  • the number of DNA fragments in the DNA fragment group of the first strand or the DNA fragment group of the second strand can also be increased or decreased according to actual needs.
  • the division of DNA strands of different lengths can be achieved by increasing or decreasing the above-mentioned DNA fragments. Specifically, whether the DNA fragment group of the first strand or the DNA fragment group of the second strand includes other DNA fragments, and the number of other DNA fragments included are determined by the sequence of the target long-chain DNA to be synthesized. Through the above design, long-chain DNAs of any of the stated lengths and desired sequences can be synthesized.
  • the nucleotide sequences of the first strand and the second strand are divided, there will be a junction between the two connected DNA fragments.
  • the adjacent DNA fragments in the first-strand DNA fragment group are The junctions are staggered from the junctions between adjacent DNA fragments in the DNA fragment group of the second strand.
  • the melting temperature (T m ) of the DNA fragments in the DNA fragment group of the first strand and the DNA fragment group of the second strand should be as close as possible, and the chain should be avoided.
  • T m melting temperature
  • the length of the 5'-end sequence of any DNA fragment in the first-strand DNA fragment group and the second-strand DNA fragment group is 4 nt or more, preferably 4-50 nt, more preferably 6-30 nt, Most preferably 10-20nt.
  • the 5' end sequence of any DNA fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the length of the 3'-end sequence of any DNA fragment in the first-strand DNA fragment group and the second-strand DNA fragment group is 4 nt or more, preferably 4-50 nt, more preferably 6-30 nt, Most preferably 10-20nt.
  • the 3' end sequence of any DNA fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the length of the continuous single-stranded DNA is 60nt or more, preferably 80nt or more, preferably 100nt or more, more preferably 60-1000nt, more preferably 80-600nt, more preferably 100-400nt, most preferably 120-nt 360nt.
  • the length of any single-stranded DNA is 60nt, 80nt, 90nt, 100nt, 120nt, 140nt, 160nt, 180nt, 200nt, 220nt, 240nt, 250nt, 260nt, 267nt, 270nt, 300nt, 320nt, 340nt, 360nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, etc.
  • the present disclosure describes a method for preparing long-chain DNA, comprising the following steps:
  • Synthesis step synthesizing the first-strand DNA fragment group and the second-strand DNA fragment group, the first-strand DNA fragment group including DNA fragment n i and DNA fragment n i+1 , the second-strand DNA fragment group
  • the group includes DNA fragment m i ; i is selected from a positive integer of 1 or more; wherein, the 5' end sequence of the DNA fragment mi and the 5' end sequence of the DNA fragment n i +1 are complementary sequences, and the 3' end sequence of the DNA fragment mi The end sequence and the 3' end sequence of the DNA fragment n i are complementary sequences;
  • Annealing step mixing the DNA fragment group of the first strand and the DNA fragment group of the second strand in the same reaction system, and annealing to form an assembly precursor of double-stranded DNA; wherein, the first strands are adjacent to each other. There is a connection port between the two DNA fragments of The junctions with the adjacent DNA fragments in the DNA fragment group of the second strand are staggered from each other;
  • Connecting step connecting the junction of any single-stranded DNA in the first strand and the second strand to obtain a double-stranded DNA assembly formed by the complementation of the continuous single-stranded DNA and the fragmented single-stranded DNA.
  • Denaturation step denaturation of the double-stranded DNA assembly to obtain continuous single-stranded DNA.
  • the method for synthesizing the DNA fragment can be a DNA synthesis method commonly used in the art, for example, chemical synthesis.
  • the short-chain DNA fragments can be prepared on a large scale by chemical synthesis, and the sequence accuracy of the DNA fragments can be guaranteed.
  • the length of any DNA fragment in the first-strand DNA fragment group and the second-strand DNA fragment group is 8-120nt, preferably 10-80nt, more preferably 15-40nt, most preferably 20-nt 30nt.
  • DNA fragments are 22nt, 24nt, 26nt, 28nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, etc. in length.
  • the length of the DNA fragment determines the difficulty and cost of its synthesis. Controlling the length of the DNA fragment at 20-30 nt can effectively reduce the difficulty of DNA fragment synthesis and control the synthesis cost.
  • the set of DNA fragments of the first strand and the set of DNA fragments of the second strand comprise modified bases at one or more positions of any of the DNA fragments.
  • modified bases are included at 1, 2, 3, 4, etc. positions of the DNA fragment.
  • the method of base modification can adopt methods commonly used in the art, for example, introducing modified bases in the process of chemically synthesizing short-chain DNA fragments. Introducing modified bases in the process of synthesizing DNA fragments can realize base modification at any site. After the DNA fragments are assembled into long-chain DNA, long-chain DNA that can precisely modify bases at any site can be obtained.
  • the following modification methods are abbreviations of commonly used modification methods. For specific modification methods, please refer to References [24-29] .
  • the modification of the base at any position in the DNA fragment can be selected from m 6 A, ⁇ , m 1 A, m 5 A, ms 2 i 6 A, i 6 A, m 3 C, m 5 C, ac 4 C, m 7 G, m2, 2G, m 2 G, m 1 G, Q, m 5 U, mcm 5 U, ncm 5 U, ncm 5 Um, D, mcm 5 s 2 U, Inosine ( I), hm 5 C, s 4 U, s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or derivatives thereof.
  • the set of DNA fragments of the first strand and the set of DNA fragments of the second strand comprise modified deoxyribose sugars at one or more positions of any of the DNA fragments.
  • modified deoxyribose sugars are included at 1, 2, 3, 4, etc. positions of the DNA fragment.
  • the method of deoxyribose modification can adopt the method commonly used in the art, for example, introducing modified deoxyribose in the process of chemically synthesizing short-chain DNA fragments.
  • the introduction of modified deoxyribose in the process of synthesizing DNA fragments can realize the modification of deoxyribose at any site. After the DNA fragments are assembled into long-chain DNA, long-chain DNA that can be precisely modified by deoxyribose at any site can be obtained. .
  • the modification mode of deoxyribose at any position in the DNA fragment can be selected from LNA, 2'-OMe, 3'-OMeU, vmoe, 2'-F, 2'-OBn (2'-O- benzyl group) or its derivatives.
  • the set of DNA fragments of the first strand and the set of DNA fragments of the second strand comprise modified phosphodiester bonds at one or more positions in any of the set of DNA fragments of the first strand, the phosphodiester bonds being formed in the short A strand of DNA between two adjacent deoxyribonucleotides.
  • modified phosphodiester linkages are included at 1, 2, 3, 4, etc. positions of the DNA fragment.
  • the method of phosphodiester bond modification can adopt methods commonly used in the art, for example, introducing modified phosphodiester bonds in the process of chemically synthesizing short-chain DNA fragments.
  • the introduction of modified phosphodiester bonds in the process of synthesizing DNA fragments can realize the modification of phosphodiester bonds at any site. Precisely modified long strands of DNA.
  • the modification mode of the phosphodiester bond at any position in the DNA fragment can be selected from phosphorothioate (PS).
  • modifications to base, deoxyribose, and phosphodiester linkages should avoid base, deoxyribose, and phosphodiester linkages immediately adjacent to the junction position to avoid first-strand or second-strand Modifications at the junction may have an effect on the junction ligation in the subsequent assembly precursor of double-stranded DNA.
  • the modified long-chain DNA By modifying at least one of the base, ribose and phosphodiester bonds at any one or more sites in the DNA fragment, and applying the modified DNA fragment to the synthesis of long-chain DNA in the present disclosure, the The precise modification of any site in the long-chain DNA effectively solves the problem that it is difficult to synthesize long-chain DNA with precise modification at a specific site in the current field.
  • the modified long-chain DNA has improved biological properties such as stability and immunogenicity, and has a wide range of uses in the field of biomedicine.
  • any DNA fragment in the set of DNA fragments of the first strand comprises a phosphate group at the 5' terminal, and a hydroxyl group at the 3' terminal.
  • the 5' end of DNA fragment ni contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of DNA fragment ni+1 contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of DNA fragment ni+2 contains a phosphate group.
  • the ' end contains a phosphate group, and the 3' end contains a hydroxyl group; the 5' end of the DNA fragment n i+3 contains a phosphate group, and the 3' end contains a hydroxyl group; the 5' end of the DNA fragment n i+4 contains a phosphate group, 3
  • the 'terminal contains a hydroxyl group; by connecting the 5' phosphate groups and the 3' hydroxyl groups on both sides of the connection port into a phosphodiester bond, the connection of the connection port in the first strand can be realized, thereby obtaining a continuous single-stranded DNA (No. One strand) is an assembly of double-stranded DNA formed complementary to fragmented single-stranded DNA (second strand).
  • any DNA fragment in the set of DNA fragments of the second strand comprises a 5' terminal phosphate group, and a 3' hydroxyl group.
  • the 5' end of DNA fragment mi contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of DNA fragment mi+1 contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of DNA fragment mi+2 contains a phosphate group.
  • the 'end contains a phosphate group and the 3'end contains a hydroxyl group
  • the 5'end of the DNA fragment mi+3 contains a phosphate group and the 3'end contains a hydroxyl group.
  • the first and second strands can be assembled.
  • the junctions in the two strands are ligated to obtain an assembly comprising double-stranded DNA formed by complementation of the continuous single-stranded DNA (second strand) and the fragmented single-stranded DNA (first strand).
  • the method for introducing the phosphate group at the 5' end of the DNA fragment can adopt a modification method commonly used in the art, for example, a phosphate group can be directly introduced at the 5' end of the DNA fragment during the synthesis of the DNA fragment; Alternatively, the phosphate group is modified at the 5' end of the DNA fragment by kinase treatment on the DNA fragment without the introduction of the phosphate group.
  • the 5' phosphate group and the 3' hydroxyl group are added to the DNA fragments of the target single-stranded DNA in the first and second strands, so that only the target single-stranded DNA is connected in the connecting step.
  • the DNA strand complementary to the target single-stranded DNA is still a fragmented DNA chain, which effectively overcomes the difficulty of denaturation of double-stranded long-stranded DNA, and the purity and recovery rate of single-stranded DNA after denaturation. Low problem, and avoid the need to digest, cut and other processing of the complementary strand in the subsequent recovery of the target single-stranded DNA.
  • DNA molecules contain 4 different deoxyribonucleotides, which are adenine deoxyribonucleotide (A), guanine deoxyribonucleotide (G), and cytosine deoxyribonucleoside depending on the type of base. acid (C) and thymidine (T).
  • the bases can be connected to each other through hydrogen bonds, and hydrogen bonds can be formed between A and T, and C and G, respectively.
  • the precise complementary pairing ability between base pairs enables the two reverse DNA single strands whose sequences are complementary to each other to form an accurate double-stranded structure by hydrogen bonding.
  • the first-strand DNA fragment group and the second-strand DNA fragment group are dissolved in the same solvent, and the two are thoroughly mixed to obtain a reaction system for preparing a double-stranded DNA assembly precursor.
  • the present disclosure does not specifically limit the specific solvent, which can be a polar solvent commonly used in the art, such as water and the like.
  • the molar ratio of any two DNA fragments in the DNA fragment group of the first strand and the DNA fragment group of the second strand is 1:(0.1-10), preferably 1:( 0.5-1), most preferably 1:1.
  • the molar ratio of any two DNA fragments is 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:2, 1:4, 1:6, 1:8, etc.
  • the assembly efficiency of short-chain DNA fragments can be improved.
  • the pH of the reaction system is set to 3-11, preferably pH 4-10, more preferably pH 5-9, and most preferably pH 3-11.
  • pH 6-8 preferably, the pH of the reaction system is 6, 7, 8, 9, and the like.
  • the temperature is lowered to form the assembly of the double-stranded DNA. body precursor;
  • the incubation temperature is any temperature of 0-100°C, preferably any temperature of 10-85°C, more preferably any temperature of 20-65°C, and the incubation time is any desired time;
  • the speed of the cooling can be any speed, and the temperature can be any temperature at which the DNA fragments in the reaction system are hybridized to form an assembly precursor of double-stranded DNA.
  • the 5' phosphate groups and the 3' hydroxyl groups on both sides of the connection port are connected as phosphodiester bonds.
  • the ligation method can be enzymatic ligation by T4 DNA ligase, Taq DNA ligase, PfU ligase, etc., or a chemical ligation method. After the ligation port is connected, a complete double-stranded DNA assembly is obtained to realize the preparation of long-chain DNA.
  • the preparation method of the present disclosure further includes a denaturation step.
  • the denaturation step the double-stranded DNA assembly is subjected to denaturation treatment to obtain continuous single-stranded DNA dispersed in the reaction system, that is, the target long-stranded DNA.
  • the method of denaturation treatment can be a method commonly used in the art for melting double-stranded DNA to form single-stranded DNA.
  • the continuous single-stranded DNA and the fragmented single-stranded DNA can be unwound by treating at a temperature of 70 °C, or in a solution containing 7M urea at a constant temperature of 50 °C, and the continuous single-stranded DNA can be dispersed. in the reaction system.
  • the preparation method of the present disclosure also includes a purification step.
  • the purification step is to purify the continuous single-stranded DNA from the reaction system.
  • the present disclosure does not specifically limit the purification method, and can be various methods for efficiently recovering DNA from the reaction system.
  • the long-chain DNA without other substances obtained after the purification step can be further applied in different fields such as clinical, drug research and development, and biological research.
  • the preparation method in the present disclosure has all the advantages of conventional DNA chemical synthesis methods (including no need for template strands, precise site modification, etc.)
  • the difficulty of chemical synthesis is reduced, and the high accuracy, high yield and site-specific modification ability of short-chain DNA fragments prepared by chemical synthesis are retained.
  • the DNA fragments that can be easily prepared by chemical synthesis are reassembled into the double-stranded DNA assembly precursor of the target structure in a specific order through the self-assembly ability of nucleic acid, and any one of the assemblies is assembled by enzymatic or chemical ligation.
  • the ligation ports of the single-stranded DNA are reconnected through phosphodiester bonds to obtain a double-stranded DNA assembly formed by the complementarity of the continuous single-stranded DNA and the fragmented single-stranded DNA.
  • For the assembly of double-stranded DNA only simple denaturation is required to obtain single-stranded target long-chain DNA. Since the chemical synthesis process can realize the precise modification of any position of the initial short-chain DNA fragment (except the bases immediately adjacent to the two sides of the junction), the obtained target long-chain DNA also has the ability to be accurately modified at almost any position. characteristics.
  • a second aspect of the present disclosure provides a long-chain DNA, which is prepared by the method of the first aspect and is a single-stranded long-chain DNA.
  • the long-chain DNA of the present disclosure can achieve accurate modification at any site, and the long single-stranded DNA itself has no sequence dependence on the modification.
  • the application in the field provides the basis.
  • experimental techniques and experimental methods used in the present embodiment are conventional technical methods, such as experimental methods that do not specify specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer. Materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • the DNA sequences used in the examples were purchased from Qingke Company without additional treatment before use. All experimental water used ultrapure water produced by 18.2M ⁇ cm Millipore Company. T4 DNA ligase and 10X ligase buffer were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. All other chemical reagents were of analytical grade. Fragment analyzer model: Fragment Capillary electrophoresis system (Siboquan Technology (Hong Kong) Co., Ltd.).
  • the long-strand DNA with the first and second strands of 100 nt and 80 nt respectively (referred to as DNA100/80) is used as the target long-strand, and the long-strand is cut into nine short-strand DNAs of 20 nt in length.
  • D-n1, D-n2, D-n3, D-n4, D-n5 are the DNA fragments for the synthesis of the first strand
  • D-m1, D-m2, D-m3 and D-m4 are for the synthesis of the second strand DNA fragments.
  • Example 3 In order to synthesize longer DNA sequences, the system was extended in Example 3 to increase the amount of short-chain DNA. Specific steps are as follows:
  • the first and second strands are respectively 252nt and 240nt long DNA (called DNA 252/240) and 315nt and 300nt long DNA (called DNA315/300) as the target long chain.
  • the DNA fragments divided by the first strand (252nt) include: D1-n1 to D1-n11; the DNA fragments divided by the second strand (240nt) include: D1-m1 to D1-m12.
  • DNA 315/300 the DNA fragments divided by the first strand (315nt) include: D2-n1 to D2-n11; the DNA fragments divided by the second strand (300nt) include: D2-m1 to D2-m10 .
  • Table 2 The specific selection of the aforementioned sequences is shown in Table 2.
  • Fig. 6 shows the characterization result of non-denaturing polyacrylamide gel electrophoresis
  • Fig. 6b shows the characterization result of the fragment analyzer. It can be seen from Figure 6 that using this method, DNA252/240 and DNA315/300 can be efficiently assembled in one pot, which provides a guarantee for the subsequent synthesis of long sequences.

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Abstract

L'invention concerne un procédé de préparation d'un ADN à longue chaîne, comprenant : une étape de synthèse, une étape de recuit et une étape de ligature. Dans le procédé, des fragments d'ADN dans un premier brin et un second brin sont conçus pour réaliser une modification chimique de n'importe quel site précis dans un ADN à chaîne longue, de telle sorte que l'ADN à longue chaîne peut obtenir des propriétés telles qu'une stabilité accrue et une immunogénicité améliorée. Selon le procédé, un ensemble ADN double brin formé par complémentation d'un ADN monocaténaire continu et d'un ADN monocaténaire fragmenté peut être obtenu, et un ADN monocaténaire à longue chaîne peut être obtenu uniquement par simple dénaturation de l'ensemble ADN double brin, ce qui simplifie efficacement les étapes de synthèse d'ADN à longue chaîne présentes, améliore l'efficacité de synthèse, et facilite la préparation industrielle à grande échelle.
PCT/CN2021/098430 2021-02-10 2021-06-04 Procédé de préparation d'adn à chaîne longue modifié dirigé sur site WO2022170707A1 (fr)

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