WO2023230991A1 - 新型闭合线性双链dna的体外制备技术 - Google Patents

新型闭合线性双链dna的体外制备技术 Download PDF

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WO2023230991A1
WO2023230991A1 PCT/CN2022/096863 CN2022096863W WO2023230991A1 WO 2023230991 A1 WO2023230991 A1 WO 2023230991A1 CN 2022096863 W CN2022096863 W CN 2022096863W WO 2023230991 A1 WO2023230991 A1 WO 2023230991A1
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dna
sequence
double
stranded dna
target
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PCT/CN2022/096863
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赵晟
唐传青
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宜明(苏州)细胞生物科技有限公司
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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
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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the invention belongs to the field of nucleic acid preparation, and particularly relates to a method for efficiently preparing a stable novel closed linear double-stranded deoxyribonucleic acid (DNA) in vitro using a restriction endonuclease that can produce non-palindromic sticky ends.
  • DNA deoxyribonucleic acid
  • DNA-based gene vectors are mostly modified from various microbial plasmids and inevitably contain microbial DNA sequences and resistance gene sequences that are unrelated to the target gene. When these products are widely used, these sequences will enter the host (such as humans) or be integrated into the genome of pathogenic bacteria or hosts through parallel gene transfer, thus causing unpredictable potential safety issues.
  • traditional DNA vaccines are constructed using circular plasmids grown in bacteria (usually Escherichia coli). The origin of replication sequence, prokaryotic promoter, prokaryotic antibiotic gene sequence, etc. from the bacteria in the plasmid will all enter the human body, possibly through parallel genes. Transferred and integrated into the genomes of humans and pathogenic bacteria, creating safety hazards that cannot be ignored and affecting vaccine safety. Therefore, when producing DNA vaccines, it is necessary to avoid introducing other additional sequences of non-antigenic genes as much as possible.
  • dbDNA doggybone DNA
  • the present invention designs a new type of stable closed linear double-stranded DNA, named Golden Cudgel DNA (abbreviated as GC-DNA), and involves the use of specific restriction endonucleases that can produce non-palindromic structure sticky ends.
  • Enzymatic method for preparing GC-DNA Compared with traditional enzymatic digestion/ligation methods, the method has the advantages of being more efficient and easy to implement, with high yield, less by-products, and is more convenient for industrial-scale amplification and production.
  • the GC-DNA preparation method can accurately remove excess sequences from bacteria directly from conventional plasmids through simple steps, producing high purity, low mutation rate at the plasmid level, resistance to nucleases, and retaining the original DNA on the plasmid. Modified and highly stable closed linear double-stranded DNA.
  • the invention provides a method for preparing closed linear double-stranded DNA, comprising:
  • a double-stranded DNA such as a plasmid or PCR product, etc.
  • a double-stranded DNA such as a plasmid or PCR product, etc.
  • the double-stranded DNA is cut at both ends to produce a linear target double-stranded DNA with protruding sticky ends of a non-palindromic structure at both ends, and the two sticky ends cannot complementary pair with each other, wherein for each enzyme cleavage site, its cleavage The site is located between its recognition sequence and the target sequence
  • stem-loop DNA Providing stem-loop DNA, the stem-loop DNA respectively having protruding sticky ends complementary to the protruding sticky ends of the non-palindromic structure at both ends of the linear target double-stranded DNA sequence, wherein the stem-loop DNA
  • One or more nucleotides in the cyclic part are modified, for example, by phosphorothioate, carboxyl, amino, amide, aldimine, ketal, acetal, ester, ether, Disulfide or aldehyde modification, preferably phosphorothioate modification
  • the target sequence, the linear target double-stranded DNA, and the closed linear double-stranded DNA do not contain the restriction within Dicer recognition sequence.
  • the invention provides a closed linear double-stranded DNA obtained or obtainable by the method of the invention, wherein one or more nucleotides in the circular portion at the end of the linear closed double-stranded DNA are modified
  • modified by phosphorothioate, carboxyl group, amino, amide group, aldimine group, ketal group, acetal group, ester group, ether group, disulfide group or aldehyde group preferably modified by phosphorothioate. of.
  • the invention provides a plasmid DNA comprising a given sequence or a target sequence and a double-stranded DNA sequence having restriction endonuclease cleavage sites at both ends of the given sequence or target sequence, wherein for The cutting site of each enzyme cutting site is located between its recognition sequence and a given sequence or target sequence, wherein the restriction endonuclease can cut the plasmid DNA at both ends of the given sequence or target sequence, producing two Linear double-stranded DNA with protruding sticky ends that are non-palindromic structures, and the two sticky ends cannot complement each other.
  • the plasmid contains the nucleotide sequence shown in SEQ ID NO: 1.
  • the present invention can produce closed linear double-stranded DNA in a cell-free environment. It can not only replace traditional plasmid DNA, but also can be used as a DNA drug to express gene products in vivo, and has broad application prospects. Similarly, because the closed linear double-stranded DNA produced by the present invention has high safety, good stability, and simple process, it is a good choice for the source of DNA in DNA vaccines; in addition, because the closed linear double-stranded DNA produced by the present invention has a simple structure, And to express the target protein, it can be applied in the fields of gene editing therapy or cell therapy.
  • Figure 1 Schematic diagram of the GC-DNA production process.
  • FIG. 2 Plasmid DNA map (A) and composition model (B).
  • the plasmid DNA used to prepare GC-DNA in the examples was obtained through artificial gene synthesis and subcloning (Anhui General Biotechnology Co., Ltd.), that is, a BsmBI restriction site was added between both sides of the target DNA sequence and the bacterial sequence, and these two The sequence of the BsmBI restriction site is reversed.
  • the DNA sequence of interest includes the promoter (CMV promoter), reporter gene (CDS region of green fluorescent protein GFP (SEQ ID NO: 3)) and polyA tail.
  • FIG. 3 BsmBI restriction site design.
  • A is the BsmBI recognition sequence and cleavage site.
  • B is the DNA linker sequence involved in the example and a schematic diagram of the product after enzyme digestion.
  • C is a diagram of the connection mode of different fragments in the connection system, where a is the connection mode of the stem-loop linker and the target fragment. Due to the complementary base pairing, it can be connected into a closed linear double-stranded DNA; b is the connection mode of the stem-loop linker and the non-target fragment.
  • c is the self-ligation model of the target fragment, and self-ligation cannot occur because the ends of the target fragment are not complementary to each other
  • d is the connection model between the target fragment and the non-target fragment, because in the connection system BsmBI enzyme activity is maintained, and the fragments are cleaved after ligation, so no ligation product can be formed.
  • Figure 4 Schematic diagram of stem-loop joint formation. After high-temperature annealing, the adapter primer can form a stem-loop structure, with a free end at the 5' end of the adapter. Asterisks indicate phosphorothioate modifications that increase the resistance of the terminal cyclic single-stranded region to enzymatic cleavage.
  • Figure 5 GC-DNA formation and identification. After linear DNA is ligated to stem-loop adapters, the sample is purified. The samples were identified using Exo III exonuclease, and the successfully connected DNA will not be degraded by Exo III. Sample 1 is plasmid DNA (positive control), sample 2 is enzyme digestion product (negative control), and sample 3 is the product of linear DNA and stem-loop adapter connection. The arrow indicates the location of the target band.
  • Figure 6 Effect of the molar ratio of linear DNA to stem-loop linker in the connection system on the formation of GC-DNA.
  • Sample 1 is plasmid DNA (positive control)
  • sample 2 is enzyme digestion product (negative control)
  • samples 3-6 are ligation products of different ligation systems (the molar ratios of linear DNA and stem-loop adapter are approximately 1:1.5 and 1: respectively. 3, 1:6, 1:12).
  • the arrow indicates the location of the target band.
  • Figure 7 Production of GC-DNA by PCR method.
  • A is the gel electrophoresis picture of the PCR product;
  • B is the gel electrophoresis picture of GC-DNA identified by Exo III.
  • the arrow part is the destination strip.
  • FIG. 8 High performance liquid chromatography (HPLC) purification of GC-DNA.
  • HPLC high performance liquid chromatography
  • the GC-DNA produced by the PCR method is purified through a molecular sieve chromatographic column. Since different band sizes are different, the time required to pass through the chromatographic column is different, and DNA of different sizes can be separated.
  • A is the HPLC peak diagram and the gel electrophoresis diagram of different collected samples.
  • B is the concentration detection results of different samples collected.
  • Figure 9 GC-DNA functional verification.
  • the picture shows the picture after 48 hours of transfection.
  • the left side is the fluorescence field picture of the control group, and the right side is the fluorescence field picture of the closed linear double-stranded DNA.
  • Figure 10 GC-DNA stability verification, showing the relative content of the DNA sequence of the GFP gene in cells 7 days after transfection compared with 3 days after transfection.
  • DNA sequence, nucleotide sequence, and nucleic acid sequence are used interchangeably and refer to the sequence of nucleotides in a deoxyribonucleic acid molecule.
  • references to nucleic acid sequences in this document are from left to right in the 5' to 3' direction; references to amino acid sequences are from left to right in the amino to carboxyl direction.
  • references to nucleic acid sequences in this document are from left to right in the 5' to 3' direction; references to amino acid sequences are from left to right in the amino to carboxyl direction.
  • references to nucleic acid sequences in this document are from left to right in the 5' to 3' direction; references to amino acid sequences are from left to right in the amino to carboxyl direction.
  • references to nucleic acid sequences in this document are from left to right in the 5' to 3' direction; references to amino acid sequences are from left to right in the amino to carboxyl direction.
  • the present invention utilizes restriction endonucleases (including but not limited to Type IIS restriction endonucleases) that can produce non-palindromic sticky ends to design sticky ends that connect target fragments and stem-loop joints, effectively avoiding by-products. .
  • restriction endonucleases including but not limited to Type IIS restriction endonucleases
  • ligation with the stem-loop linker can effectively form a closed end and effectively suppress the generation of incorrect ligation products.
  • the present invention adds phosphorothioate modification to the nucleotides of the cyclic part of the stem-loop linker to inhibit the degradation of the cyclic part by nucleases and make the formed closed linear Double-stranded DNA is more stable in the body and improves gene expression efficiency.
  • the invention provides a method for preparing closed linear double-stranded DNA that is resistant to nuclease degradation, comprising:
  • a double-stranded DNA such as plasmid or PCR product, etc.
  • the double-stranded DNA is cleaved end-to-end to produce a linear target double-stranded DNA with protruding sticky ends of a non-palindromic structure at both ends, and the two sticky ends cannot complement each other.
  • the cleavage site The point is between its recognition sequence and the target sequence,
  • stem-loop DNA Provide stem-loop DNA, the stem-loop DNA having protruding sticky ends respectively complementary to the protruding sticky ends of the non-palindromic structure at both ends of the linear target double-stranded DNA sequence, wherein the stem-loop
  • One or more nucleotides in the circular portion of the DNA are modified, for example, by phosphorothioate, carboxyl, amino, amide, aldimine, ketal, acetal, ester, ether groups , disulfide group or aldehyde group modification, preferably phosphorothioate modification,
  • the target sequence, the linear target double-stranded DNA, and the closed linear double-stranded DNA do not contain the restriction within Dicer recognition sequence.
  • DNA refers to deoxyribonucleic acid, the backbone of which consists of deoxyribonucleotides (deoxyadenine nucleotides (A), deoxyguanine nucleotides (G), deoxycytosine nucleotides (C), Deoxythymine nucleotides (T)) are linear or cyclic polymers connected by 3',5'-phosphodiester bonds.
  • the DNA may contain suitable modifications known in the art, such as methylation, thiolation, and the like.
  • double-stranded DNA refers to a DNA molecule in which at least part of the two strands (eg, except for the sticky ends and single-stranded circular portions at the ends), preferably the entire sequence, are complementary base pairs.
  • linear double-stranded DNA refers to double-stranded DNA with a 5' end and a 3' end, which is not a circular DNA molecule. When linear double-stranded DNA is completely denatured, two single strands of DNA will form. Linear double-stranded DNA may contain single-stranded circular portions as part of it.
  • closed linear double-stranded DNA refers to a DNA molecule in which the 5' and 3' ends of the linear double-stranded DNA are closed by a single-stranded circular part, that is, the "Golden Cudgel” structure ( Figure 1 shown).
  • the sequence of the closed linear double-stranded DNA includes a single-stranded circular portion - a linear double-stranded DNA sequence portion - a single-stranded circular portion.
  • one or more nucleotides of the single-chain cyclic moiety are modified, for example, by phosphorothioate, carboxyl, amino, amide, aldimine, ketal, acetal, Modified with ester group, ether group, disulfide group or aldehyde group, preferably with phosphorothioate modification.
  • phosphorothioate carboxyl, amino, amide, aldimine, ketal, acetal
  • Modified with ester group, ether group, disulfide group or aldehyde group preferably with phosphorothioate modification.
  • non-complementary pairing with each other means that two nucleic acid sequences cannot base pair to form double-stranded DNA.
  • the target fragment is shown in Figure 3B.
  • target DNA and target sequence are used interchangeably and refer to any DNA sequence having a DNA sequence of interest, including, for example, protein coding sequences, promoter sequences, regulatory sequences, transgene insert sequences wait.
  • a linear target double-stranded DNA with two sticky ends may have two 5' protruding sticky ends, two 3' protruding sticky ends, or one 5' protruding sticky end and one 3' protruding sticky end. .
  • sticky ends refer to single-stranded unpaired sequences at the (5' or 3') end of a DNA molecule.
  • a 5' overhanging sticky end refers to a single-stranded unpaired nucleotide sequence at the 5' end of a DNA molecule; a 3' overhanging sticky end refers to a single-stranded unpaired nucleotide sequence at the 3' end of a DNA molecule. sequence.
  • the length of the sticky end may be 4-10 nucleotides, preferably 4, 5, or 6 nucleotides.
  • the sticky end mainly consists of G and C, for example, the G+C content accounts for more than 50% of the total nucleotide content in the sticky end, such as at least 60%, at least 70%, at least 80%, at least 90% or even 100%.
  • the number of G+Cs is 6, 7, 8, 9, or 10, or when the length of the sticky end is 6 nucleotides, the number of G+Cs is 4, 5, or 6, or when the length of the sticky end is 4 nucleotides, the number of G+C is 3 or 4.
  • the sticky ends consist only of G and C. In a particularly preferred embodiment, the sticky end sequence is CGGC.
  • the linear target double-stranded DNA preferably has two identical 5' protruding sticky ends or two identical 3' protruding sticky ends. Since a DNA molecule with two identical protruding sticky ends is formed, it is only necessary to provide a stem-loop DNA linker with complementary sticky ends. After ligation, the two ends of the linear target double-stranded DNA can be closed to form a closed linear double-stranded DNA. Stranded DNA molecules.
  • the protruding sticky ends of the linear target double-stranded DNA can be generated by digestion of the DNA with specific restriction enzymes.
  • Restriction endonucleases are enzymes that recognize specific nucleotide sequences and cut DNA.
  • Recognition sequence refers to the specific sequence in double-stranded DNA that restriction endonuclease first recognizes before catalyzing DNA cleavage, and then the enzyme cuts DNA at the corresponding specific site (cutting site).
  • the cleavage site of a restriction enzyme includes a recognition sequence and a cleavage site.
  • the recognition sequence and the cleavage site may overlap or be independent, depending on the type of restriction enzyme.
  • the cleavage sites of restriction endonucleases are known in the art, namely recognition sequences and cleavage sites. For example, see Wil A M Loenen et al., "Highlights of the DNA cutters: a short history of the restriction enzymes", Nucleic Acids Res. ,Jan 2014:42(1):3-19.doi:10.1093/nar/gkt990.
  • the cleavage site between the recognition sequence and the target sequence means that the end of the restriction enzyme recognition site away from the target sequence needs to be further away from the target sequence than the end of the cleavage site away from the target sequence, thereby cutting The side close to the target sequence will not contain or be missing part of the recognition site.
  • restriction endonucleases useful in the present invention are those capable of generating non-palindromic structure protruding sticky ends, such as 5' protruding sticky ends or 3' protruding sticky ends, and the restriction endonucleases are
  • the end of the Dicer recognition site away from the target sequence needs to be further away from the target sequence than the end of the cutting site away from the target sequence. Therefore, after cutting, the side close to the target sequence will not contain or lack part of the recognition site, and the recognition site will not It will be restored after being connected to the stem loop.
  • Restriction enzymes that can be used in the present invention include, but are not limited to, BsmBI, BaeI, BbsI, BspQI, FspEI, I-CeuI, etc. It is preferred that the length of the sticky end produced is no less than 4 bases and the G/C content is no less than 50%.
  • Restriction enzymes described herein include, but are not limited to, Type IIS restriction enzymes that recognize contiguous asymmetric sequences and cleave DNA beyond the recognition site.
  • the restriction enzyme is selected from Type IIS restriction enzymes, such as BsmBI.
  • a palindrome or palindrome refers to a DNA sequence that has the same sequence as its complement when read in the same direction (e.g., 5' to 3' direction).
  • 5’-ACCTAGGT-3’ is a palindrome sequence or palindrome structure.
  • the linear target double-stranded DNA has two protruding sticky ends that are identical but not complementary to each other (non-palindromic structure), such as two identical non-palindromic 5' protruding sticky ends. Or two identical non-palindromic 3' overhanging sticky ends.
  • DNA with two identical non-palindromic 5' protruding sticky ends means that the double-stranded DNA has a 5' protruding sticky end at both ends, which are in the same direction (for example, from 5' to 3' direction) when reading, the sequence of the protruding part has the same nucleic acid sequence, for example, as shown in the target fragment in Figure 3B.
  • DNA with two identical non-palindromic 3' protruding sticky ends means that the double-stranded DNA has a 3' protruding sticky end at both ends, which are in the same direction (for example, both from 3' When reading in the 5' direction), the sequence of the overhang has the same nucleic acid sequence.
  • the sticky end sequences at both ends of the linear target double-stranded DNA are not palindromic sequences. Therefore, in the generated linear target double-stranded DNA, one of the two identical sticky ends is not complementary to the other, and thus cannot spontaneously and effectively form double-stranded DNA, and will not be preferentially connected by T4-DNA polymerase to produce by-products.
  • stem-loop DNA refers to a DNA molecule having a single-stranded circular portion, a double-stranded portion, and sticky ends.
  • the stem-loop DNA of the present invention has only one sticky end.
  • the stem-loop DNA of the present invention has only one single-stranded circular part.
  • the stem-loop DNA includes a protruding sticky end, which is complementary to the protruding sticky end of the linear target double-stranded DNA of the present invention, and will not regenerate the original target after subsequent ligation with the linear target double-stranded DNA.
  • Cutting sites for special restriction endonucleases at both ends of the sequence are provided.
  • the sticky ends of stem-loop DNA can be efficiently ligated and improve GC-DNA yields by complementary pairing with the sticky ends of linear target double-stranded DNA.
  • the stem-loop DNA having a protruding sticky end complementary to the sticky end of the linear target double-stranded DNA means that the stem-loop DNA has a sticky end that can be complementary to the sticky end of the linear target double-stranded DNA to form a double strand. Since stem-loop DNA has only one sticky end, if the linear target double-stranded DNA has two different sticky ends, you need to provide two types of stem-loop DNA, each with two different sticky ends from the linear target double-stranded DNA. Complementary sticky ends.
  • the sticky end of the stem-loop DNA and the sticky end of the linear target double-stranded DNA complementary to it are both 5' protruding sticky ends or 3' protruding sticky ends. That is, if the sticky ends of the linear target double-stranded DNA If the sticky end of the linear target double-stranded DNA is a 3'-protruding sticky end, then the sticky end of the stem-loop DNA that is complementary to it is also a 5'-protruding sticky end. The sticky ends of circular DNA are also 3' protruding sticky ends.
  • the sticky end of the target DNA and the sticky end of the stem-loop DNA can form a double strand, which will facilitate the connection between the stem-loop DNA and the target DNA and improve the efficiency of forming a closed linear target double-stranded DNA molecule.
  • the stem-loop DNA length may be 20-50 nucleotides, preferably 20-40 nucleotides, more preferably 20-30 nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides.
  • the loop portion in the stem-loop DNA may be 5-20 nucleotide residues in length, preferably 5-15 nucleotide residues, and more preferably 10-15 nucleosides.
  • Acid residues include, for example, 10, 11, 12, 13, 14, and 15 nucleotide residues.
  • nucleotides can be modified to resist nuclease degradation, including but not limited to phosphorothioate, carboxyl, amino, amide, aldimine, ketal, acetal, ester, ether groups , disulfide group and aldehyde group and other modifications.
  • phosphorothioate modification refers to replacing a non-bridging oxygen atom in the phosphate bond of a nucleotide with a sulfur atom.
  • the nucleotide sequence is more stable and resistant to nucleases. Degradation, thereby enhancing the in vivo stability of nucleic acid molecules. It is known in the art how to carry out thiomodification, for example by ordering a specified number and position of modified oligonucleotide adapters directly from a primer synthesis company.
  • nucleotides in the circular portion of the stem-loop DNA are modified.
  • one or more, or even all, nucleotides in the circular portion of the stem-loop DNA are modified, for example, phosphorothioate, carboxyl, amino, amide, aldimine, Modified with ketal group, acetal group, ester group, ether group, disulfide group or aldehyde group.
  • one or more, or even all, nucleotides in the circular portion of the stem-loop DNA are phosphorothioate modified.
  • the stem-loop DNA comprises the following nucleotide sequence:
  • gccgATCGCGAG*A*G*G*G*T*T*G*A*CTCGCGAT (SEQ ID NO: 5), where one or more, preferably all, of the nucleotides indicated by the asterisk are modified, for example It is modified with phosphorothioate.
  • Stem-loop DNA with protruding sticky ends complementary to the sticky ends of the linear target double-stranded DNA can be generated by any suitable method, including, for example, amplification (eg, PCR), synthesis, digestion with endonucleases, and the like. Generating DNA molecules with specific sequences is well known in the art.
  • ligation refers to the joining of one DNA molecule to another to form a new DNA molecule.
  • Ligation can be performed by any suitable technique known in the art, for example using T4-DNA ligase, T3-DNA ligase, T7-DNA ligase, E. coli DNA ligase, Taq DNA ligase, thermostable 5'App DNA ligation Enzymes etc.
  • Ligase enzymes for joining nucleic acid molecules are known in the art, including but not limited to T4-DNA ligase.
  • the molar ratio of linear target double-stranded DNA and stem-loop DNA can be any suitable ratio, such as 1:1 to 1:15, 1:1 to 1:14, 1:1 to 1:13, 1:1 to 1:12, 1:1 to 1:11, 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1: 7, 1:1 to 1:6, 1:2 to 1:15, 1:2 to 1:14, 1:2 to 1:13, 1:2 to 1:12, 1:2 to 1:11, 1:2 to 1:10, 1:2 to 1:9, 1:2 to 1:8, 1:2 to 1:7, 1:2 to 1:6, 1:3 to 1:15, 1: 3 to 1:14, 1:3 to 1:13, 1:3 to 1:12, 1:3 to 1:11, 1:3 to 1:10, 1:3 to 1:9, 1:3 to 1:8, 1:3 to 1:7, 1:3 to 1:6.
  • the molar ratio of linear target double-stranded DNA and stem-loop DNA at the time of ligation is 1:3 to 1:6.
  • the ligation efficiency and product purity of the closed linear double-stranded DNA are improved.
  • the linear target double-stranded DNA and the stem-loop DNA are ligated in the presence of the restriction endonuclease, which can cleave ligation by-products (such as re- Circularized plasmid DNA containing the restriction endonuclease cleavage site or re-ligated into the original PCR product of the target sequence containing the restriction endonuclease cleavage site at both ends), thereby improving the required Closes the yield of linear double-stranded DNA and suppresses the generation of ligation by-products.
  • the target sequence, the linear target double-stranded DNA, and the closed linear double-stranded DNA do not contain an enzyme cleavage site for the restriction endonuclease.
  • isolation and/or purification of the formed closed linear double-stranded DNA can be performed using any suitable method or means known in the art, such as chromatography, HPLC, precipitation, and the like.
  • an exonuclease such as Exonuclease III (Exo III)
  • the exonuclease, such as Exo III can remove non-closed linear double-stranded DNA in the post-ligation system to purify the desired closed linear double-stranded DNA.
  • the invention provides a method for preparing closed linear double-stranded DNA that is resistant to nuclease degradation, comprising:
  • stem-loop DNA having a 5' protruding sticky end that is complementary to the 5' protruding sticky end of the linear target double-stranded DNA and a 3' protruding sticky end that is complementary to the 3' protruding sticky end of the linear target double-stranded DNA.
  • 'Stem-loop DNA protruding from the sticky end wherein one or more nucleotides in the circular portion of the stem-loop DNA are modified, for example, by phosphorothioate, carboxyl, amino, amide, aldimine, ketal group, acetal group, ester group, ether group, disulfide group or aldehyde group modification, preferably phosphorothioate modification,
  • the target sequence, the linear target double-stranded DNA, and the closed linear double-stranded DNA do not contain the restriction within Dicer enzyme cleavage site.
  • the invention provides a method for preparing closed linear double-stranded DNA that is resistant to nuclease degradation, comprising:
  • stem-loop DNA each of which has a sticky end that is complementary to a sticky end of the linear target double-stranded DNA sequence, wherein one or more of the circular portions of the stem-loop DNA
  • the nucleotide is modified, for example by a phosphorothioate, carboxyl, amino, amide, aldimine, ketal, acetal, ester, ether, disulfide or aldehyde group, preferably Phosphorothioate modified,
  • the target sequence, the linear target double-stranded DNA, and the closed linear double-stranded DNA do not contain the restriction within Dicer enzyme cleavage site.
  • the linear target double-stranded DNA has two identical 5' protruding sticky ends or two identical 3' protruding sticky ends, wherein the sequence of the sticky ends is not a palindromic sequence.
  • the linear target double-stranded DNA having two protruding sticky ends that are not complementary to each other is provided as follows:
  • a double-stranded DNA (such as linear double-stranded DNA) having the complementary sequence of the cleavage site of the first restriction endonuclease, the target DNA sequence, and the cleavage site of the second restriction endonuclease, For each enzyme cleavage site, its cleavage site is located between its recognition sequence and the target sequence,
  • the method includes ligating said target DNA and stem-loop DNA in the presence of said restriction enzyme to form a closed linear double-stranded DNA.
  • complementary sequence refers to a sequence that is complementary to a given sequence in an A-T or G-C format in the opposite direction (i.e., 5'-3'/3'-5'), thereby forming a double-stranded DNA, e.g., 5' -ATGC-3' and 5'-GCAT-3' are complementary sequences to each other.
  • DNA containing the complement of a given sequence means that the complement of the given sequence is present in the 5' to 3' direction on the DNA.
  • DNA containing the complement of the sequence 5'-ATGC-3' means that the DNA contains (in the 5' to 3' direction) the sequence 5'-GCAT-3'.
  • sequences at both ends of the nucleotide sequence shown in the upper panel of Figure 3B are shown.
  • a "sticky end” is a protruding single-stranded unpaired end formed by cleavage at the cleavage site of a restriction endonuclease described herein upon recognition of a specific recognition sequence by the enzyme.
  • Those skilled in the art can design the required protruding sticky ends based on the cleavage site characteristics of restriction endonucleases known in the art.
  • the cleavage site and recognition sequence of the enzyme may be different sequences, or may be part of the recognition sequence, as long as the enzyme can form the required protruding sticky end after cutting the DNA.
  • the restriction endonuclease cleaves the DNA double strand at bases other than the recognition site (ie, the cleavage site is not within the recognition sequence), forming a sticky end, for example, selected from Type IIS As a restriction enzyme, BsmBI is particularly preferred.
  • the restriction site can be designed more flexibly to form the desired protruding sticky end.
  • the linear target double-stranded DNA produced after enzyme digestion contains as few non-target DNA sequences as possible.
  • the restriction endonuclease cleavage site is designed so that after digestion, except for the protruding sticky end sequence, the remaining sequences of the restriction endonuclease site (such as the recognition sequence) do not exist in the generated linear target doublet. in strand DNA.
  • the cleavage site of the enzyme cleavage site is placed between the recognition sequence of the enzyme cleavage site and the target sequence.
  • the double-stranded DNA should contain 5'-recognition sequence-cleavage site-target sequence or its complementary sequence-3 at the end of one strand ' or 5' - the target sequence or its complementary sequence - the complementary sequence of the cleavage site - the complementary sequence of the recognition sequence - 3', so that after digestion, at least part of the recognition sequence will not exist in the DNA fragment containing the target sequence.
  • the double-stranded DNA should contain 5'-target sequence or its complementary sequence-cutting site-recognition sequence-3 at the end of one strand.
  • ' or 5' - the complementary sequence of the recognition sequence - the complementary sequence of the cleavage site - the target sequence or its complementary sequence - 3', so that after digestion, at least part of the recognition sequence will not exist in the DNA fragment containing the target sequence.
  • a double-stranded DNA contains a target sequence and restriction enzyme cleavage sites at both ends of the target sequence, wherein at each end, the cleavage site of the restriction endonuclease is located at its The sequences between the recognition sequence and the target sequence and the two restriction sites are reversed.
  • the "two sequences are reversed" means that one of the two sequences (5'-3' direction) is located on one strand of the double-stranded DNA, and the other sequence (5'-3' direction) is on one strand of the double-stranded DNA. ' direction) is located on the complementary strand.
  • the double-stranded DNA includes on the same strand: the recognition sequence of the first restriction endonuclease - the target sequence - the complementary sequence of the cleavage site of the second restriction endonuclease.
  • the double-stranded DNA includes on the same strand: the recognition sequence of the first restriction endonuclease - the cleavage site of the first restriction endonuclease - the target sequence - the second restriction enzyme Complementary sequence of the cleavage site of the endonuclease - Complementary sequence of the recognition sequence of the second restriction endonuclease.
  • restriction endonucleases and design enzyme cutting sites based on the knowledge about restriction endonucleases known in the art, so as to form the desired protruding sticky residue after the restriction endonuclease digests the DNA. ends, and satisfy the requirement that, except for the recognition sequences in the enzyme cutting sites at both ends of the double-stranded DNA, the target sequence, linear target double-stranded DNA and closed linear double-stranded DNA of the present invention do not contain the restriction endonuclease. Identification sequence requirements.
  • the enzyme cleavage site of the BsmBI enzyme is: CGTTCCN1N2N3N4N5, where the recognition sequence is CGTTCCN1, and its cleavage site is N2N3N4N5.
  • the enzyme is 3' of N1 and the 4th downstream of the recognition sequence.
  • the 5' position of the complementary pairing base of the nucleotide base can form a sticky end with a 5' overhang of 4 nucleotides (N2N3N4N5), where N1-N5 are any nucleotide bases. Therefore, the sequence composition of the cutting site sequence N2N3N4N5 can be designed as needed to form the required protruding sticky end sequence. Therefore, restriction enzymes that cleave DNA at sites other than the recognition sequence are particularly preferred.
  • the double-stranded DNA contains a BsmBI restriction site (CGTCTCN1N2N3N4N5 (SEQ ID NO: 11) on one strand, wherein N1-N5 is any one of deoxyribonucleotides A, T, C, and G.
  • BsmBI restriction site CGTCTCN1N2N3N4N5 (SEQ ID NO: 11) on one strand, wherein N1-N5 is any one of deoxyribonucleotides A, T, C, and G.
  • the complementary sequence of the target DNA sequence and the restriction site of another restriction endonuclease for example, the complementary sequence of the BsmBI restriction site: N5'N4'N3'N2'N1'GAGACG (SEQ ID NO: 12), where N1'-N5' are the nucleotide bases paired with N1-N5 respectively
  • N2-N5 At least 3 or all 4 are selected from G and C.
  • the double-stranded DNA comprises CGTCTCN1N2N3N4N5 (SEQ ID NO: 11), the target DNA sequence and N5'N4'N3'N2'N1'GAGACG (SEQ ID NO: 12) on one strand
  • N1-N5 are any of deoxyribonucleotides A, T, C, and G
  • N1'-N5' are the nucleotide bases paired with N1-N5 respectively.
  • the condition is that N2N3N4N5 is not a palindrome sequence, and N2- is preferred. At least 3 or all 4 of N5 are selected from G and C.
  • the double-stranded DNA comprises CGTTCTCGGC (SEQ ID NO: 4), the target DNA sequence and GCCGAGAGACG (SEQ ID NO: 13) on one strand.
  • any suitable method known in the art can be used to generate the DNA (such as linear double-stranded DNA) molecules having specific nucleotide sequences at both ends, including but not limited to amplification (such as PCR), molecular ligation, etc. .
  • primers can be designed to include specific sequences and complementary sequences specific to the upstream and downstream of the DNA sequence of interest, thereby generating the two ends through amplification techniques such as PCR DNA having the specific sequence respectively.
  • the linear target double-stranded DNA with sticky ends having non-palindromic structures at both ends and unable to complement each other is provided as follows:
  • step (3) Digest the PCR amplification product obtained in step (2) with the restriction enzyme to generate the linear target double-stranded DNA with sticky ends that have non-palindromic structures at both ends and cannot be complementary to each other.
  • the target double-stranded DNA can be cloned into a plasmid with two restriction sites, such that the target sequence has one restriction site at each end.
  • a linear target double-stranded DNA with non-palindromic protruding sticky ends at both ends can be produced, and the two sticky ends cannot complement each other.
  • the linear target double-stranded DNA with sticky ends having non-palindromic structures at both ends and unable to complement each other is provided as follows:
  • a template plasmid DNA that contains at least one enzyme cleavage site on both sides of a given sequence.
  • its cleavage site is located between its recognition sequence and the given sequence.
  • plasmid DNA is digested with a restriction enzyme that recognizes the restriction site, a DNA fragment with protruding sticky ends with non-palindromic structures at both ends can be produced, and the two sticky ends cannot be complementary to each other,
  • the restriction site does not exist elsewhere in the plasmid sequence
  • step (3) Digest the plasmid obtained in step (2) with a restriction endonuclease that recognizes the enzyme cleavage site to obtain a linear target double-stranded DNA with sticky ends protruding from a non-palindromic structure at both ends, and these two The sticky ends cannot complement each other.
  • sequences of the two sticky ends may be the same or different, and are preferably the same.
  • the above step (3) obtains a linear target double-stranded DNA with two 5' protruding sticky ends that are non-palindromic and not complementary to each other. More preferably, a linear target double-stranded DNA with two identical Non-palindromic 5' overhangs of the sticky ends of target DNA.
  • the above step (3) obtains a linear target double-stranded DNA with two 3' protruding sticky ends that are non-palindromic and not complementary to each other. More preferably, a linear target double-stranded DNA with two identical The non-palindromic 3' overhang of the sticky end of the target DNA.
  • a plasmid is a circular double-stranded DNA vector capable of replicating in an organism that contains an origin of replication, a cloning site, and optionally a selectable marker, such as an antibiotic resistance gene.
  • the plasmids described herein contain two enzyme cleavage sites capable of the restriction endonuclease, wherein the recognition sequences of the two enzyme cleavage sites are located on different strands of double-stranded DNA, whereby the endonuclease cleaves
  • the plasmid DNA can obtain a target DNA fragment having two protruding sticky ends that are not complementary to each other.
  • the plasmid contains the BsmBI restriction site CGTTCCN 1 N 2 N 3 N 4 N 5 and its complementary sequence on the same chain, where N 1 -N 5 are deoxyribonucleotides A, T , C, G, and at least 3 or all 4 of N 2 -N 5 are selected from G and C, provided that N 2 N 3 N 4 N 5 is not a palindrome sequence.
  • the plasmid contains the DNA sequence CGTTCTCGGC (SEQ ID NO: 4) and its complement on the same strand.
  • the plasmid comprises the nucleotide sequence shown in SEQ ID NO: 1.
  • cloning the target double-stranded DNA into a plasmid refers to inserting the target DNA sequence at the desired position, that is, the target double-stranded DNA, by suitable means known in the art (for example, using a restriction enzyme). Both ends of the sequence are connected to the enzyme cutting sites respectively.
  • digestion is the incubation of the enzyme with the DNA molecule under conditions suitable for the restriction enzyme to cleave the DNA nucleic acid molecule, such that the restriction enzyme recognizes the restriction enzyme recognition sequence in the DNA molecule and cleaves the DNA accordingly. molecular. Digestion conditions required for specific enzymes are known in the art, such as pH, temperature, presence or absence of auxiliary ions such as magnesium ions, etc.
  • the restriction endonuclease is simultaneously present in the reaction system. This can effectively prevent the cut partial sequence from being reconnected back to the original sequence. Therefore, no purification is required after enzyme digestion, and the ligation system can be directly prepared for the ligation reaction.
  • the invention provides linear closed DNA obtained or obtainable by the method of the invention.
  • the linear closed DNA sequence comprises a stem-loop portion - a target double-stranded DNA sequence portion - a stem-loop portion, wherein one or more nucleotides of the stem-loop portion are modified, e.g., by sulfide Modified by phosphoric acid ester, carboxyl group, amino group, amide group, aldimine group, ketal group, acetal group, ester group, ether group, disulfide group or aldehyde group, preferably phosphorothioate modification.
  • Linear closed DNA obtained by the methods described herein is resistant to nuclease degradation and has improved stability.
  • the invention provides a plasmid DNA comprising a first restriction endonuclease cleavage site, a given sequence or a target sequence and a second restriction endonuclease cleavage site, wherein for Each restriction endonuclease site is located between its recognition sequence and a given sequence or target sequence, wherein the plasmid is digested by the first and second restriction enzymes to produce a strand having A linear double-stranded DNA segment with a non-palindromic protruding sticky end, and the two sticky ends cannot complement each other.
  • the plasmid is digested with the restriction enzyme to produce a sticky end with two 5' protruding ends or two 3' protruding sticky ends, especially two identical 5' protruding sticky ends or Two identical DNA fragments with 3' overhanging sticky ends, and the sequence of the overhanging sticky ends is not a palindromic sequence.
  • a given sequence refers to the DNA sequence that is replaced by the target DNA when the target DNA is cloned into a plasmid, which may be any suitable DNA sequence, including, for example, prokaryotic sequences, multiple cloning sites, and the like.
  • the first restriction endonuclease and the second restriction endonuclease may be the same or different, and are preferably the same.
  • the plasmid comprises the DNA sequence CGTTCN 1 N 2 N 3 N 4 N 5 and its complementary sequence on the same strand, wherein N 1 -N 5 are deoxyribonucleotides A, T, C, G any, and at least 3 or all 4 of N 2 -N 5 are selected from G and C, provided that N 2 N 3 N 4 N 5 is not a palindrome sequence, preferably, N 2 N 3 N 4 N 5 for CGGC.
  • the plasmid contains the DNA sequence CGTTCTCGGC (SEQ ID NO: 4) and its complement on the same strand.
  • the plasmid comprises the nucleotide sequence shown in SEQ ID NO: 1.
  • the closed linear double-stranded DNA production method disclosed in the present invention is simple and fast, avoids mismatching or mutation of the target sequence, and ensures sequence accuracy.
  • the present invention proves that the closed linear double-stranded DNA produced by the method of the present invention has the normal function of expressing proteins. Therefore, the closed linear double-stranded DNA produced according to the method of the present invention can replace traditional plasmid DNA and has broad application prospects.
  • the method of the present invention is simple and the closed linear GC-DNA produced has high safety and good stability, it can be used In the field of DNA vaccines; in addition, GC-DNA can also be widely used in fields such as gene editing, gene therapy or cell therapy.
  • the term “about” refers to a range of values including the specific value that one skilled in the art would reasonably consider to be similar to the specific value. In some embodiments, the term “about” means within standard error using measurements generally accepted in the art. In some embodiments, about refers to +/-10% of the specified value.
  • Ranges disclosed herein should be considered to specifically disclose all possible subranges and individual values within such ranges. For example, a description of a range from 1 to 6 should be deemed to have expressly disclosed subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc. , as well as single numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the scope.
  • the main materials involved in the examples include plasmid DNA (SEQ ID NO: 1) and adapter primer (SEQ ID NO: 5) synthesized by Anhui General Biotechnology Company, BsmBI fast endonuclease and buffer (ThermoFisher Scientific Company of the United States), T4 -DNA ligase and buffer (Nanjing Novezan Biotechnology Co., Ltd.), Exo III exonuclease and buffer (ThermoFisher Scientific Company, USA), 50 ⁇ TAE buffer (Shanghai Sangon Bioengineering Company), nucleic acid dyes (Shanghai Sangon Bioengineering Company), agarose (Shanghai Sangon Bioengineering Company), DNA Ladder (ThermoFisher Scientific Company of the United States), 293T cells (ATCC Center of the United States), polyethylenimine (PEI, Sigma-Aldrich Company of the United States) wait.
  • SEQ ID NO: 1 plasmid DNA
  • SEQ ID NO: 5
  • the linear DNA used in this example was obtained from template plasmid DNA by restriction endonuclease method.
  • the template plasmid was obtained through gene synthesis and subcloning by Anhui General Biotechnology Company.
  • the plasmid contains target sequences such as promoter, reporter gene and polyA tail, and the restriction sites of Type IIS restriction endonuclease BsmBI are added to both ends. Click to separate the sequence of interest from the bacterial sequence ( Figure 2B).
  • the template plasmid DNA map is shown in Figure 2, and the nucleotide sequence is shown in SEQ IN NO: 1.
  • the specific process is:
  • the primer sequence (SEQ IN NO: 5) was synthesized from Anhui General Biotechnology Company.
  • the primers undergo high-temperature annealing to eventually form a stem-loop linker, and the end of the stem-loop linker has a sticky end that can bind to the linear target DNA (shown in Figure 4).
  • phosphorothioate modification is added to the nucleotides in the loop region during the synthesis of primers. This modification can prevent the single-stranded loop portion from being degraded by nucleases.
  • the primers are diluted with ribozyme-free water (1 ⁇ M) for annealing reaction, denatured by keeping at 95°C for 5 to 10 minutes, and then slowly lowered to maximize the formation of stem-loop junctions. The final temperature can be lowered to 4°C.
  • T4-DNA ligase 5 ⁇ l/100 ⁇ l reaction system
  • ligation buffer Najing Novezan Biotechnology Co., Ltd.
  • enzyme digestion into the linear DNA (10 ⁇ g/100 ⁇ l reaction system) mixture obtained later. React at 16°C for 16 hours, or incubate overnight.
  • the obtained ligation product is purified by ethanol precipitation or a commercial purification and recovery kit. Take 0.3 to 1 ⁇ g of the purified product, add appropriate Exo III exonuclease (ThermoFisher Scientific, USA) and its buffer, mix evenly, and react at 37°C for 15 minutes. Normal plasmid DNA was used as the positive control group, and samples obtained through enzyme digestion or PCR were used as the negative control group. After the reaction, the samples were detected by gel electrophoresis to maintain consistent sample loading quality.
  • Exo III exonuclease ThermoFisher Scientific, USA
  • the molar ratio of linear target DNA to stem-loop linker in the ligation system will affect the ligation efficiency, that is, the GC-DNA yield.
  • the present invention compares the differences in connection products with different molar ratios.
  • the specific implementation process is: mix the linear target DNA and the stem-loop adapter at different molar ratios ranging from 1:1 to 1:15, then add appropriate T4-DNA ligase and ligation buffer; react at 16°C for 16 hours, or Direct overnight incubation. After the reaction, the obtained ligation product is purified by ethanol precipitation or a commercial purification and recovery kit. Then take 0.3 to 1 ⁇ g of the purified product, add appropriate Exo III exonuclease (ThermoFisher Scientific, USA) and its buffer, mix evenly, and react at 37°C for 15 minutes. At the same time, normal plasmid DNA was used as the positive control group, and samples obtained through enzyme digestion or PCR were used as the negative control group. After the reaction, the samples were detected by gel electrophoresis to maintain consistent sample loading quality.
  • the band brightness of the ligation product in the 1:3 to 1:6 molar ratio range changed less, indicating that closed linear double-stranded DNA (GC-DNA) was present in the product.
  • the ratio is higher.
  • PCR primers (SEQ IN NO: 6 and 7) based on the target fragment sequence in the template plasmid.
  • the primers contain the recognition sequence of the restriction endonuclease BsmBI.
  • PCR high-fidelity enzyme (Nanjing Novozan Biotechnology Co., Ltd.) was used to prepare the PCR system according to the instructions and perform the PCR reaction.
  • the PCR product (SEQ ID NO: 8) can be detected by gel electrophoresis. The results are shown in Figure 7A.
  • the PCR product also contains two non-specific bands. This is related to the specificity of the primers. The generation of non-specific bands can be reduced by changing the primers or increasing the annealing temperature.
  • PCR product is then purified and recovered through gel electrophoresis or a PCR purification kit.
  • GC-DNA was prepared by enzymatic digestion and ligation methods as described in Example 1 for subsequent operations, that is, the PCR product was digested with restriction endonuclease BsmBI and ligated with a stem-loop linker. After ligation is completed, the product is identified using Exo III exonuclease. The results are shown in Figure 7B. After the Exo III exonuclease reaction, the target band exists in GC-DNA, indicating that GC-DNA can be obtained by PCR.
  • the sample is treated with Exo III exonuclease or further purified by passing it through a chromatography column.
  • Exo III exonuclease or further purified by passing it through a chromatography column.
  • the in vitro ligation products were purified using a molecular sieve chromatography column (Suzhou Saifen Technology Co., Ltd.) according to the different band sizes.
  • HPLC high-performance liquid chromatography
  • molecular sieve chromatography column Operate according to the instructions of high-performance liquid chromatography (HPLC) and molecular sieve chromatography column.
  • the mobile phase is Tris-HCl buffer (pH 7.5).
  • concentration detection or gel electrophoresis detection can be performed.
  • Figure 8 After HPLC purification, the molecular sieve chromatographic column used can clearly separate the small bands.
  • the sample purification effect is related to the sensitivity of the chromatographic column. By replacing a suitable molecular sieve chromatographic column, it is possible to obtain higher purity closed linear double-stranded DNA.
  • the obtained GC-DNA was transfected into cells, and whether the reporter gene (green fluorescent protein GFP (SEQ ID NO: 3)) was expressed under a fluorescence microscope was observed.
  • the reporter gene green fluorescent protein GFP (SEQ ID NO: 3)
  • the specific implementation process is as follows: 293T cells are passaged and inoculated one day before transfection.
  • the cell culture and passage methods are carried out according to the specific methods in "Cell Culture Technology” (edited by Lan Rong and Zhou Zhenhui).
  • the reagents involved are all from commercial products.
  • the prepared GC-DNA (1 ⁇ g) is introduced into the cells through transfection.
  • the transfection method can be selected such as lipofection, calcium transfection, PEI transfection or electroporation. 48 hours after transfection, GFP expression in cells can be observed.
  • the present invention conducts cell transfection with the obtained GC-DNA, culture the cells for 7 days, detects the relative content of DNA sequences in the cells through fluorescence quantitative PCR, and compares it with the transfected plasmid DNA and linear Groups of DNA are compared (by testing the relative content of the GFP gene sequence contained in each group, SEQ ID NO: 3).
  • the specific implementation process is as follows: 293T cells are passaged and inoculated one day before transfection.
  • the cell culture and passage methods are carried out according to the specific methods in "Cell Culture Technology" (Lan Rong).
  • the reagents involved are all from commercial products.
  • the transfection method can be selected such as lipofectamine transfection, calcium transfection, and PEI transfection. dyeing or electroporation.
  • Cells are collected after 3 and 7 days of cell culture. The cell pellet is dissolved in a buffer and stored.
  • the buffer can be PBS or TE buffer.
  • the collected cell samples were frozen and thawed three times, then centrifuged and the supernatant was collected. Fluorescence quantitative PCR detection was performed according to the instructions of the fluorescence quantification kit (Norvizan).
  • the exogenous DNA that initially enters the cells through transfection will be gradually degraded within the cells and will also be continuously diluted through cell division. Seven days after transfection, the exogenous DNA content in the same number of cells was significantly lower than that after 3 days of transfection (the relative ratio between the two was less than 100%), but the exogenous DNA content in the GC-DNA group after 7 days of transfection was 3 days after transfection. The percentage of days after was significantly higher than that of the circular plasmid group and the linear DNA group (results shown in Figure 10). This shows that the stability of the GC-DNA produced by the present invention in cells is significantly higher than that of circular plasmids and linear DNA.
  • Plasmid DNA nucleotide sequence SEQ IN NO:1

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Abstract

一种制备闭合线性双链DNA的方法，包括：(a)提供一条含目标序列和至少两个限制性内切酶的酶切位点的双链DNA，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，(b)用限制性内切酶消化双链DNA，产生一条两端均具有非回文粘端的目标DNA，且这两个粘端不能彼此互补配对，(c)提供茎环DNA，其分别具有与目标DNA的粘端互补配对的粘端，其中所述茎环DNA的环状部分中的核苷酸是修饰的，(d)连接目标DNA和茎环DNA形成闭合线性双链DNA。

Description

新型闭合线性双链DNA的体外制备技术 技术领域
本发明属于核酸制备领域，特别涉及使用可产生非回文结构粘端的限制性内切酶体外高效制备一种稳定的新型闭合线性双链脱氧核糖核酸(DNA)的方法。
背景技术
传统基于DNA的基因载体大多由各类微生物质粒改造而来，不可避免的含有与目的基因无关的微生物DNA序列和抗性基因序列。当这些产品被广泛使用时，这些序列就会进入宿主(如人)体内，或者通过平行基因转移整合进致病细菌或宿主的基因组，从而带来难以估计的潜在安全性问题。例如，传统DNA疫苗是以环状质粒在细菌(通常为大肠杆菌)中生长形式来构建，质粒中来自细菌的复制原点序列，原核启动子，原核抗生素基因序列等都会进入人体，可能通过平行基因转移整合到人和致病细菌的基因组，产生不可忽视的安全隐患，影响疫苗安全性。因而在生产DNA疫苗时，需要尽可能避免引入非抗原基因的其他附加序列。
这些多余的序列可以通过常规的酶切线性化后再纯化去除，但开环的线性化目标DNA的稳定性也会下降。也可以把开环的线性目标DNA再连接成两端闭合的DNA来增加稳定性，然而现有的常规酶切和连接技术由于会产生大量的连接副产物，实际获得目标闭合线性双链DNA的生产效率很低，分子量和带电量相似的目标DNA和副产物DNA也难以高效的分离纯化，很难实现放大的规模化工业生产。
最近几年，出现了一种生产不含细菌序列的DNA载体的技术，其中DNA只包含所需最小序列，包括抗原序列、启动子和polyA尾，这种新型DNA是DNA双链末端由磷酸二酯键连接闭合的线性DNA，被称为doggybone DNA(dbDNA)。与传统质粒DNA相比，dbDNA依赖于体外滚环复制的酶促反应，产生不含细菌序列的末端闭合线性双链DNA，末端的闭合性使之在细胞内较之开放末端的常规线性DNA更稳定，半衰期更长。然而，这种以滚环复制形式进行扩增的过程涉及体外聚合酶反应，大量的体外扩增反应很容易出现碱基错配或突变，从而引入新的安全性和产物一致性问题。对于长片段抗原序列，扩增过程需要的时间更长，这种不确定性的积累也会更明显。通过细菌制备传统质粒时，由于细菌内天然具有复杂的错配/突变修复系统，突变率远低于体外扩增反应。此外，质粒DNA在细菌中进行复制的过程中，DNA可以被甲基化和其他修饰，会进一步提高DNA的稳定性和表达效率。然而，这些修饰过程在体外滚环复制的扩增系统中也是不具备的，因而缺乏甲基化等修饰的dbDNA在DNA的稳定性及表达效率上也可能存在难以避免的短板。因此，本领域需要新的设计和方法来简便和快速的制备不含细菌序列的DNA载体。
发明内容
本发明设计了一种新型稳定的闭合线性双链DNA，命名为金箍棒DNA(Golden Cudgel DNA，缩写为GC-DNA)，并涉及使用特定的可产生非回文结构粘端的限制性内切酶来制备GC-DNA的方法。所述方法较之传统的酶切/连接方法具有更高效易行，产量高，副产物少，更便于进行工业级放大和生产的优势。与dbDNA制备方法相比，所述GC-DNA制备方法可以直接从常规质粒通过简单的步骤准确去掉来自细菌的多余序列，制备高纯度、质粒级别的低突变率、抗核酸酶、保留质粒上原有修饰且高稳定性的闭合线性双链DNA。
在一个方面，本发明提供了一种制备闭合线性双链DNA的方法，包括：
(a)提供一条含目标序列和分别在目标序列两端的限制性内切酶的酶切位点的双链DNA(如质粒或PCR产物等)，其中所述限制性内切酶可在目标序列两端切割该双链DNA，产生两端均具有非回文结构的突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，
(b)用所述限制性内切酶消化所述双链DNA，产生一条两端均具有非回文结构的突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
(c)提供茎环DNA，所述茎环DNA分别具有与所述线性目标双链DNA序列两端的非回文结构突出的粘端分别互补配对的突出的粘端，其中，所述茎环DNA的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的
(d)连接(b)获得的线性目标双链DNA和(c)的茎环DNA以形成闭合线性双链DNA，优选在存在所述限制性内切酶的情况下进行连接，和
(e)任选地，分离和/或纯化形成的闭合线性双链DNA，
其中，除在(a)的双链DNA中酶切位点中的识别序列外，所述目标序列、所述线性目标双链DNA、所述闭合线性双链DNA均不含有所述限制性内切酶的识别序列。
在一个方面，本发明提供了通过本发明所述方法获得或可获得的闭合线性双链DNA，其中在所述线性闭合双链DNA末端的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的。
在一个方面，本发明提供了一种质粒DNA，其包含给定序列或目标序列和在给定序列或目标序列两端具有限制性内切酶的酶切位点的双链DNA序列，其中对于每个酶切位点，其切割位点位于其识别序列与给定序列或目标序列之间，其中所述限制性内切酶可在给定序列或目标序列两端切割该质粒DNA，产生两端均具有非回文结构的突出的粘端的线性双链DNA，且这两个粘端不能彼此互补配对。优选地，所述质粒包含SEQ ID NO：1所示的核苷酸序列。
本发明可在无细胞环境下生产闭合线性双链DNA，不仅可替代传统质粒 DNA，而且可作为DNA药物进行体内表达基因产物，具有广泛的应用前景。同样，由于本发明生产的闭合线性双链DNA安全性高、稳定性好，且流程简单，是DNA疫苗中DNA来源的一个良好选择；另外，由于本发明生产的闭合线性双链DNA结构简单，且表达目的蛋白，可应用于基因编辑治疗或细胞治疗领域。
附图说明
图1：GC-DNA生产流程示意图。
图2：质粒DNA图谱(A)及组成模式图(B)。实施例中制备GC-DNA的质粒DNA是通过人工基因合成和亚克隆获得(安徽通用生物公司)，即在目标DNA序列两侧与细菌序列之间分别添加一个BsmBI酶切位点而且这两个BsmBI酶切位点的序列是反向的。感兴趣的DNA序列包含启动子(CMV启动子)、报告基因(绿色荧光蛋白GFP(SEQ ID NO：3)的CDS区)及polyA尾。
图3：BsmBI酶切位点设计。A为BsmBI识别序列及切割位点。B为实施例中涉及的DNA接头序列，及酶切后产物模式图。C为连接体系中不同片段连接模式图，其中a为茎环接头与目的片段连接模式图，由于碱基互补配对，可连接为闭合线性双链DNA；b为茎环接头与非目的片段连接模式图，由于碱基不互补配对，不能连接；c为目的片段自连模式图，由于目的片段末端不互补配对，不能发生自连；d为目的片段与非目的片段连接模式图，由于连接体系中保持了BsmBI酶活性，片段连接后又被切开，所以不能形成连接产物。
图4：茎环接头形成模式图。接头引物经过高温退火后，可形成茎环结构，接头5’末端有游离末端。星号表示硫代磷酸酯修饰，可提高末端环状单链区的抗酶解能力。
图5：GC-DNA形成及鉴定。线性DNA与茎环接头连接之后，样品进行纯化。用Exo III核酸外切酶对样品经行鉴定，连接成功的DNA不会被Exo III降解。样品1为质粒DNA(阳性对照)，样品2为酶切产物(阴性对照)，样品3为线性DNA与茎环接头连接产物。箭头为目的条带位置。
图6：连接体系中线性DNA与茎环接头摩尔比对GC-DNA形成影响。样品1为质粒DNA(阳性对照)，样品2为酶切产物(阴性对照)，样品3-6为不同连接体系的连接产物(线性DNA与茎环接头摩尔比分别约为1:1.5、1:3、1:6、1:12)。箭头为目的条带位置。
图7：通过PCR法生产GC-DNA。A为PCR产物凝胶电泳图；B为Exo III鉴定GC-DNA的凝胶电泳图。箭头部分为目的条带。
图8：高效液相色谱(HPLC)纯化GC-DNA。将PCR法生产获得的GC-DNA经过分子筛色谱柱进行纯化，由于不同条带大小不同，经过色谱柱时需要的时间有差异，可将不同大小DNA进行分离。A为HPLC峰图及收集的不同样品的凝胶电泳图。B为收集的不同样品的浓度检测结果。
图9：GC-DNA功能验证。图为转染48小时后的图片，左侧为对照组的荧光视野图，右侧为闭合线性双链DNA的荧光视野图。
图10：GC-DNA稳定性验证，显示与转染细胞3天后相比，转染细胞7天后的细胞内GFP基因的DNA序列相对含量。
具体实施方式
除非另有定义，本文所用的技术和科学术语具有本领域技术人员通常理解的含义。本发明中未作具体说明的实验方法，均根据《分子克隆实验指南》(第四版)J.萨姆布鲁克一书中具体方法进行，或者按照相关产品说明书进行。本发明中所用生物试剂，无特殊说明，均可以从商业途径获得。本领域技术人员在不偏离本发明精神的范围内可以进行多种变化、改变和替代。
本文中，DNA序列、核苷酸序列、核酸序列可以互换使用，是指脱氧核糖核酸分子的核苷酸排列顺序。
除非另有说明，本文中提及核酸序列时从左至右为5′至3′方向；提及氨基酸序列时从左至右为氨基至羧基方向。除非特别指明或者根据上下文能够确定，本文中提及DNA序列的结构或组成时均是指在双链DNA分子的一条链上。特别地，提及包含在目标序列两端的限制性内切酶的酶切位点时，这两个酶切位点可以在同一条链上，也可以分别在两条链上。
本发明利用能产生非回文结构粘端的限制性内切酶(包含但不限于Type IIS类限制性内切酶)，设计了连接目的片段和茎环接头的粘端，有效的避免了副产物。当经过限制性内切酶进行酶切并获得目的片段后，与茎环接头进行连接可以有效形成闭合末端并且能有效抑制错误连接产物的生成。此外，为进一步提高GC-DNA的稳定性，本发明在茎环接头的环状部分的核苷酸上增加了硫代磷酸酯修饰，抑制核酸酶对环状部分的降解，使形成的闭合线性双链DNA在体内更加稳定，提高基因表达效率。
在一个方面，本发明提供了一种制备能够抗核酸酶降解的闭合线性双链DNA的方法，包括：
(a)提供一条含目标序列和在目标序列两端的限制性内切酶的酶切位点的双链DNA(如质粒或PCR产物等)，其中所述限制性内切酶可在目标序列两端切割该双链DNA，产生两端均具有非回文结构的突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，
(b)用所述限制性内切酶消化所述线性目标双链DNA，产生一条两端均具有非回文结构的突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
(c)提供茎环DNA，所述茎环DNA分别具有与所述线性目标双链DNA序列两端的非回文结构的突出的粘端分别互补配对的突出的粘端，其中，所述茎环DNA的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的，
(d)连接(b)获得的线性目标双链DNA和(c)的茎环DNA以形成闭合线性双链DNA，优选在存在所述限制性内切酶的情况下进行连接，和
(e)任选地，分离和/或纯化形成的闭合线性双链DNA，
其中，除在(a)的双链DNA中酶切位点中的识别序列外，所述目标序列、所述线性目标双链DNA、所述闭合线性双链DNA均不含有所述限制性内切酶的识别序列。
如本文所用，DNA是指脱氧核糖核酸，主链由脱氧核糖核苷酸(脱氧腺嘌呤核苷酸(A)、脱氧鸟嘌呤核苷酸(G)、脱氧胞嘧啶核苷酸(C)、脱氧胸腺嘧啶核苷酸(T))通过3’,5’-磷酸二酯键连接形成的线形或环形多聚体。DNA可以包含本领域已知的合适修饰，例如甲基化、硫代等。
如本文所用，双链DNA是指两条链至少部分(例如除末端的粘端和单链环状部分外)、优选全部序列是碱基互补配对的DNA分子。
如本文所用，线性双链DNA是指存在5’端和3’端的双链DNA，其不是环状DNA分子。当线性双链DNA完全变性时，将形成两条DNA单链。线性双链DNA可以含有作为其一部分的单链环状部分。
如本文所用，闭合线性双链DNA是指线性双链DNA的5’端和3’端通过单链环状部分被闭合的DNA分子，即“金箍棒(Golden Cudgel)”结构(如图1所示)。由此，所述闭合线性双链DNA的序列包含单链环状部分-线性双链DNA序列部分-单链环状部分。特别地，所述单链环状部分的一或多个核苷酸是被修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的。当该DNA完全变性时，将形成单链环状DNA。
本领域已知如何产生具有粘端的DNA分子，例如通过酶切、扩增等方法。
如本文所用，彼此不互补配对是指两个核酸序列不能通过碱基配对形成双链DNA。例如图3B中目的片段所示。
如本文所用，目标DNA和目标序列可互换使用，是指具有感兴趣的DNA序列的任何DNA序列，所述感兴趣的DNA序列例如包括蛋白质编码序列、启动子序列、调控序列、转基因插入序列等。
在本文中，具有两个粘端的线性目标双链DNA可以具有两个5’突出的粘端、两个3’突出的粘端、或者一个5’突出的粘端和一个3’突出的粘端。
如本文所用，粘端是指DNA分子(5’或3’)末端的单链未配对序列。
如本文所用，5’突出的粘端是指在DNA分子的5’端的单链未配对核苷酸序列；3’突出的粘端是指在DNA分子的3’端的单链未配对核苷酸序列。
在一个实施方案中，所述粘端的长度可为4-10个核苷酸，优选为4、5、6个核苷酸。
在一个实施方案中，所述粘端主要由G和C组成，例如G+C含量占粘端中总核苷酸含量的50％以上，例如至少60％、至少70％、至少80％、至少90％或者甚至100％。例如，粘端长度为10个核苷酸时，G+C数目为6、7、8、9或10 个，或者粘端长度为6个核苷酸时，G+C数目为4、5或6个，或者粘端长度为4个核苷酸时，G+C数目为3或4个。
在一个特别优选的实施方案中，粘端仅由G和C组成。在一个特别优选的实施方案中，粘端序列为CGGC。
在一个优选实施方案中，所述线性目标双链DNA优选具有两个相同的5’突出的粘端或者两个相同的3’突出的粘端。由于形成了具有两个相同的突出的粘端的DNA分子，因此只需提供一种具有互补粘端的茎环DNA接头，连接后即可闭合所述线性目标双链DNA的两端，形成闭合线性双链DNA分子。
在一个实施方案中，所述线性目标双链DNA的突出的粘端可由特定限制性内切酶消化DNA产生。
限制性内切酶是指能够识别特定核苷酸序列并且切割DNA的一类酶。识别序列是指限制内切酶在催化DNA切割前首先识别的双链DNA中的特异序列，之后该酶在相应的特定位点(切割位点)切割DNA。
如本文所用，限制性内切酶的酶切位点包括识别序列和切割位点，识别序列和切割位点位置上可以重叠也可以是独立的，这取决于限制性内切酶的类型。本领域已知限制性内切酶的酶切位点即识别序列和切割位点，例如参见Wil A M Loenen等，“Highlights of the DNA cutters:a short history of the restriction enzymes”，Nucleic Acids Res.,Jan 2014:42(1):3-19.doi:10.1093/nar/gkt990。因此，根据本领域已知技术知识，本领域技术人员可以选择合适的限制性内切酶，以产生本文所述两端均具有非回文结构的突出的粘端且这两个粘端不能彼此互补配对的线性目标双链DNA。
如本文所用，切割位点位于识别序列与目标序列之间是指所述限制性内切酶识别位点远离目标序列的一端需要比切割位点远离目标序列的一端离目标序列更远，从而切割后靠近目标序列一侧将不含或缺失部分识别位点。
特别地，可用于本发明的限制性内切酶是能够产生非回文结构突出的粘端例如5’突出的粘端或3’突出的粘端的限制性内切酶，而且所述限制性内切酶识别位点远离目标序列的一端需要比切割位点远离目标序列的一端离目标序列更远，从而切割后靠近目标序列一侧将不含或缺失部分识别位点，而且该识别位点不会在和茎环连接后重新恢复。可用于本发明的限制性内切酶例如包括但不限于BsmBI，BaeI，BbsI，BspQI，FspEI，I-CeuI等。优选产生的粘端长度不少于4个碱基，且G/C含量不低于50％。
本文所述限制性内切酶包括但不限于识别连续的非对称序列并且在识别位点之外切割DNA的Type IIS类限制性内切酶。在一个优选实施方案中，所述限制性内切酶选自Type IIS类限制性内切酶，例如BsmBI。
如本文所用，回文序列或回文结构是指一段DNA序列，与其互补序列在以相同方向(例如5’至3’方向)读取时具有相同的序列。例如，5’-ACCTAGGT-3’是回文序列或回文结构。
在一个优选的实施方案中，所述线性目标双链DNA具有的两个突出的粘端 相同但彼此不互补配对(非回文结构)，例如两个相同的非回文5’突出的粘端或者两个相同的非回文3’突出的粘端。
在本文中，DNA具有两个相同的非回文5’突出的粘端是指该双链DNA两端各有一个5’突出的粘端，在以相同方向(例如均从5’至3’方向)读取时，突出部分的序列具有相同的核酸序列，例如图3B中目的片段所示。同理，如本文所用，DNA具有两个相同的非回文3’突出的粘端是指该双链DNA两端各有一个3’突出的粘端，在以相同方向(例如均从3’至5’方向)读取时，突出部分的序列具有相同的核酸序列。
在本文中，所述线性目标双链DNA两端的粘端序列不是回文序列。由此，在产生的线性目标双链DNA中，两个相同粘端的一个与另一个不是互补的，从而不能自发有效的形成双链，不会优先被T4-DNA聚合酶连接起来产生副产物。
如本文所用，茎环DNA是指具有单链环状部分、双链部分和粘端的DNA分子。当茎环DNA完全变性时，形成线性单链DNA分子。本发明所述茎环DNA仅具有一个粘端。优选地，本发明所述茎环DNA仅具有一个单链环状部分。
在本文中，茎环DNA包括突出的粘端，其与本发明所述的线性目标双链DNA的突出的粘端互补，且后续与所述线性目标双链DNA连接后不会重新生成原目标序列两端的特殊限制性内切酶的酶切位点。
如本文所述，茎环DNA的粘端通过与线性目标双链DNA的粘端互补配对，可以高效连接并提高GC-DNA产量。
在本文中，具有与所述线性目标双链DNA的粘端互补的突出的粘端的茎环DNA是指茎环DNA具有的粘端能够与线性目标双链DNA的粘端互补形成双链。由于茎环DNA只有一个粘端，因此，如果线性目标双链DNA具有两个不同的粘端，则需要提供两种茎环DNA，分别具有与所述线性目标双链DNA两个不同的粘端互补的粘端。
根据DNA碱基配对原理，茎环DNA的粘端和与其互补的线性目标双链DNA的粘端均为5’突出的粘端或3’突出的粘端，即如果线性目标双链DNA的粘端是5’突出的粘端，则与其互补的茎环DNA的粘端也是5’突出的粘端，而如果线性目标双链DNA的粘端是3’突出的粘端，则与其互补的茎环DNA的粘端也是3’突出的粘端。由此，目标DNA的粘端与茎环DNA的粘端可以形成双链，从而将有助于茎环DNA与目标DNA的连接，提高形成闭合线性目标双链DNA分子的效率。
在一个实施方案中，茎环DNA长度可为20-50个核苷酸，优选为20-40个核苷酸，更优选为20-30个核苷酸，例如20、21、22、23、24、25、26、27、28、29、30个核苷酸。
在一个实施方案中，茎环DNA中的环状部分长度可为5-20个核苷酸残基，优选为含5-15个核苷酸残基，更优选为含10-15个核苷酸残基，例如含10、11、12、13、14、15个核苷酸残基。
本领域已知可以修饰核苷酸以抵抗核酸酶的降解，包含但不限于硫代磷酸酯、 羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基和醛基等修饰。例如，硫代磷酸酯修饰是指将核苷酸的磷酸键中一个非桥连的氧原子置换为硫原子，加入硫代磷酸酯键后核苷酸序列稳定性更好，能抵抗核酸酶的降解，因而可以增强核酸分子的体内稳定性。本领域已知如何进行硫代修饰，如可以通过向引物合成公司直接订购指定数目和位置的带修饰的寡核苷酸接头。
为增加茎环DNA和/或形成的闭合DNA的稳定性，茎环DNA中环状部分的核苷酸是被修饰的。在一个实施方案中，茎环DNA的环状部分中的一或多个、甚至全部的核苷酸是被修饰的，例如是硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰的。优选地，所述茎环DNA的环状部分中的一或多个、甚至全部的核苷酸是硫代磷酸酯修饰的。
在一个实施方案中，茎环DNA包含如下核苷酸序列：
gccgATCGCGAG*A*G*G*G*T*T*G*A*CTCGCGAT(SEQ ID NO：5)，其中星号表示的核苷酸中的一或多个、优选全部是被修饰的，例如是硫代磷酸酯修饰的。
具有与线性目标双链DNA粘端互补的突出的粘端的茎环DNA可以以任何合适的方法产生，包括例如扩增(如PCR)、合成、经内切酶消化等。产生具有特定序列的DNA分子是本领域熟知的。
如本文所用，连接是指将一个DNA分子与另一个DNA分子结合形成新的DNA分子。连接可以通过本领域已知的任何合适技术进行，例如使用T4-DNA连接酶、T3-DNA连接酶、T7-DNA连接酶、大肠杆菌DNA连接酶、Taq DNA连接酶、热稳定5’AppDNA连接酶等。本领域已知用于连接核酸分子的连接酶，包括但不限于T4-DNA连接酶。
在一个实施方案中，在连接时，线性目标双链DNA和茎环DNA的摩尔比例可以是任何合适的比例，例如1:1至1:15，1:1至1:14，1:1至1:13，1:1至1:12，1:1至1:11，1:1至1:10，1:1至1:9，1:1至1:8，1:1至1:7，1:1至1:6，1:2至1:15，1:2至1:14，1:2至1:13，1:2至1:12，1:2至1:11，1:2至1:10，1:2至1:9，1:2至1:8，1:2至1:7，1:2至1:6，1:3至1:15，1:3至1:14，1:3至1:13，1:3至1:12，1:3至1:11，1:3至1:10，1:3至1:9，1:3至1:8，1:3至1:7，1:3至1:6。在一个实施方案中，在连接时，线性目标双链DNA和茎环DNA的摩尔比例为1:3至1:6，在此范围内，出人意料地，闭合线性双链DNA的连接效率和产物纯度明显优于其他比例范围。
在一个优选的实施方案中，在存在所述限制性内切酶的情况下，连接所述线性目标双链DNA和茎环DNA，其中所述限制性内切酶可以切割连接副产物(如重新环化的含所述限制性内切酶酶切位点的质粒DNA或重新连接成的两端含所述限制性内切酶酶切位点的目标序列的原始PCR产物)，从而提高所需闭合线性双链DNA的产量和抑制连接副产物的产生。优选地，所述目标序列内、所述线性目标双链DNA内、所述闭合线性双链DNA内均不含有所述限制性内切酶的酶切位点。
如本文所用，分离和/或纯化形成的闭合线性双链DNA可以采用本领域已知的任何合适方法或手段进行，例如层析、HPLC、沉淀等。在一个实施方案中，可以使用核酸外切酶，例如Exonuclease III(Exo III)。所述核酸外切酶，例如Exo III能够去除连接后体系中的非闭合线性双链DNA，以纯化所需的闭合线性双链DNA。
在一个实施方案中，本发明提供了一种制备能够抗核酸酶降解的闭合线性双链DNA的方法，包括：
(a)提供一条含目标序列和分别在目标序列两端的限制性内切酶的酶切位点的双链DNA，其中所述限制性内切酶可在目标序列两端切割该双链DNA，产生具有非回文结构的5’突出的粘端和3’突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，
(b)用所述限制性内切酶消化所述线性目标双链DNA，产生一条具有非回文结构的粘端的5’突出的粘端和3’突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
(c)提供具有与所述线性目标双链DNA的5’突出粘端互补配对的5’突出粘端的茎环DNA和具有与所述线性目标双链DNA的3’突出粘端互补配对的3’突出粘端的茎环DNA，其中所述茎环DNA的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的，
(d)连接所述线性目标双链DNA和茎环DNA以形成闭合线性双链DNA，优选在存在所述限制性内切酶的情况下进行连接，和
(e)任选地，分离和/或纯化形成的闭合线性双链DNA，
其中，除在(a)的双链DNA中酶切位点中的识别序列外，所述目标序列、所述线性目标双链DNA、所述闭合线性双链DNA均不含有所述限制性内切酶的酶切位点。
在一个实施方案中，本发明提供了一种制备能够抗核酸酶降解的闭合线性双链DNA的方法，包括：
(a)提供一条含目标序列和分别在目标序列两端的限制性内切酶的酶切位点的双链DNA，其中所述限制性内切酶可在目标序列两端切割该双链DNA，产生具有非回文结构的两个5’突出的粘端或两个3’突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，
(b)用所述限制性内切酶消化所述线性目标双链DNA，产生具有非回文结构的两个5’突出的粘端或两个3’突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
(c)提供茎环DNA，所述茎环DNA分别具有与所述线性目标双链DNA序列的粘端互补配对的粘端，其中，所述茎环DNA的环状部分中的一或多个核 苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的，
(d)连接(b)获得的所述线性目标双链DNA和(c)的茎环DNA以形成闭合线性双链DNA，优选在存在所述限制性内切酶的情况下进行连接，和
(d)任选地，分离和/或纯化形成的闭合线性双链DNA，
其中，除在(a)的双链DNA中酶切位点中的识别序列外，所述目标序列、所述线性目标双链DNA、所述闭合线性双链DNA均不含有所述限制性内切酶的酶切位点。
在一个优选的实施方案中，所述线性目标双链DNA具有的两个相同的5’突出的粘端或两个相同的3’突出的粘端，其中所述粘端的序列不是回文序列。
在一个实施方案中，如下提供所述具有彼此不互补配对的两个突出的粘端的线性目标双链DNA：
(i)提供一条具有第一限制性内切酶的酶切位点、目标DNA序列、第二限制性内切酶的酶切位点的互补序列的双链DNA(例如线性双链DNA)，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，
(ii)用所述第一限制性内切酶和第二限制性内切酶消化所述双链DNA，产生一条两端均具有非回文结构粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，其中，除所述第一和第二酶切位点中的识别序列外，所述双链DNA、所述线性目标双链DNA和/或形成的闭合线性双链DNA中不含有所述第一和第二限制性内切酶的识别序列。
优选地，所述方法包括在存在所述限制性内切酶的情况下连接所述目标DNA和茎环DNA以形成闭合线性双链DNA。
如本文所用，“互补序列”是指与给定序列能够以相反方向(即5’-3’/3’-5’)以A-T或G-C形式互补，从而形成双链DNA的序列，例如5’-ATGC-3’和5’-GCAT-3’彼此是互补序列。
本文中，提及DNA含有一个给定序列的互补序列是指所述给定序列的互补序列在所述DNA上以5’至3’方向存在。例如，DNA含有序列5’-ATGC-3’的互补序列是指所述DNA(以5’至3’方向)含有序列5’-GCAT-3’。例如，图3B上图所示的核苷酸序列的两端序列所示。
如本文所用，“粘端”是在本文所述限制性内切酶识别特定识别序列后，在所述酶的切割位点处切割，形成的突出的单链未配对末端。本领域技术人员根据本领域已知的限制性内切酶的酶切位点特性，可以设计所需的突出的粘端。所述酶的切割位点和识别序列可以是不同的序列，也可以是识别序列的一部分，只要酶切割DNA后能够形成所需的突出的粘端即可。在一个特别优选的实施方案中，所述限制性内切酶在识别位点以外的碱基处切割DNA双链(即切割位点不在识别序列内)，形成粘端，例如选自Type IIS类限制性内切酶的酶，特别优选BsmBI。由此，可以更加灵活设计酶切位点，以形成所需的突出的粘端。
为尽量少的在产生的所需DNA中引入多余序列，在酶切之后产生的线性目 标双链DNA含有非目标DNA序列越少越好。为此，设计限制性内切酶的酶切位点，从而在酶切后，除形成突出的粘端序列外，其余酶切位点的序列(例如识别序列)不存在于产生的线性目标双链DNA中。为此，将酶切位点的切割位点置于酶切位点的识别序列和目标序列之间。由此，在用所述酶消化后，至少部分识别序列不存在于获得的线性目标双链DNA中。
对于切割位点位于识别序列下游(3’端)的限制性内切酶而言，双链DNA在一条链的末端应包含5’-识别序列-切割位点-目标序列或其互补序列-3’或5’-目标序列或其互补序列-切割位点的互补序列-识别序列的互补序列-3’，由此酶切后，至少部分识别序列不会存在于包含目标序列的DNA片段中。
对于切割位点位于识别序列上游(5’端)的限制性内切酶而言，双链DNA在一条链的末端应包含5’-目标序列或其互补序列-切割位点-识别序列-3’或5’-识别序列的互补序列-切割位点的互补序列-目标序列或其互补序列-3’，由此酶切后，至少部分识别序列不会存在于包含目标序列的DNA片段中。
因此，在一个实施方案中，提供一条含目标序列和在目标序列两端的限制性内切酶的酶切位点的双链DNA，其中在每个末端，酶切位点的切割位点位于其识别序列和目标序列之间并且两个酶切位点的序列是反向的。
本文中，所述“两个序列是反向的”是指两个序列中的一个序列(5’-3’方向)位于双链DNA中的一条链上，而另一个序列(5’-3’方向)位于互补链上。
在一个实施方案中，所述双链DNA在同一条链上包含：第一限制性内切酶的识别序列-目标序列-第二限制性内切酶的切割位点的互补序列。在一个优选的实施方案中，所述双链DNA在同一条链上包含：第一限制性内切酶的识别序列-第一限制性内切酶的切割位点-目标序列-第二限制性内切酶的切割位点的互补序列-第二限制性内切酶的识别序列的互补序列。如此，酶消化后，识别序列不再保留在所需的线性目标双链DNA片段上。
本领域技术人员可以根据本领域已知关于限制性内切酶的知识选择合适的限制性内切酶并且设计酶切位点，从而在限制性内切酶消化DNA后形成所需的突出的粘端，并满足除在双链DNA两端的酶切位点中的识别序列外，本发明所述目标序列、线性目标双链DNA和闭合线性双链DNA均不含有所述限制性内切酶的识别序列的要求。
以BsmBI酶为例，如图3A所示，BsmBI酶的酶切位点为：CGTCTCN1N2N3N4N5，其中识别序列为CGTCTCN1，其切割位点为N2N3N4N5，该酶在N1的3’和识别序列下游第4个核苷酸碱基的互补配对碱基的5’位置，从而可以形成具有4个核苷酸的5’突出的粘端(N2N3N4N5)，其中N1-N5为任意核苷酸碱基。由此，可以根据需要设计切割位点序列N2N3N4N5的序列组成，从而形成所需的突出的粘端序列。因此，特别优选这种在识别序列之外的位点切割DNA的限制性内切酶。
在一个实施方案中，所述双链DNA在一条链上包含BsmBI酶切位点(CGTCTCN1N2N3N4N5(SEQ ID NO：11)，其中N1-N5为脱氧核糖核苷酸A、 T、C、G任一)、目标DNA序列和另一限制性内切酶的酶切位点的互补序列(例如所述BsmBI酶切位点的互补序列：N5’N4’N3’N2’N1’GAGACG(SEQ ID NO：12)，其中N1’-N5’分别为与N1-N5配对的核苷酸碱基)，条件是产生的两个突出的粘端是非回文结构的且不能彼此互补配对，优选N2-N5中至少3个或所有4个选自G和C。
在一个优选的实施方案中，所述双链DNA在一条链上包含CGTCTCN1N2N3N4N5(SEQ ID NO：11)、目标DNA序列和N5’N4’N3’N2’N1’GAGACG(SEQ ID NO：12)，其中N1-N5为脱氧核糖核苷酸A、T、C、G任一，N1’-N5’分别为与N1-N5配对的核苷酸碱基，条件是N2N3N4N5不是回文序列，优选N2-N5中至少3个或所有4个选自G和C。
在进一步优选的实施方案中，所述双链DNA在一条链上包含CGTCTCTCGGC(SEQ ID NO：4)、目标DNA序列和GCCGAGAGACG(SEQ ID NO：13)。
可以使用本领域已知的任何合适的方法产生所述的两端分别具有特定核苷酸序列的DNA(例如线性双链DNA)分子，包括例如但不限于扩增(例如PCR)、分子连接等。
以开环双链DNA或闭合双链DNA作为模板DNA，其中可以设计引物，以包括特定序列和特异于感兴趣的DNA序列上下游的互补序列，从而通过扩增技术例如PCR产生所述两端分别具有所述特定序列的DNA。
在一个实施方案中，如下提供所述两端均具有非回文结构且不能彼此互补配对的粘端的线性目标双链DNA：
(1)提供感兴趣的双链DNA，
(2)提供一对引物，该对引物分别包含限制性内切酶的酶切位点及感兴趣的DNA序列上游序列或下游序列，以感兴趣的DNA序列为模板，进行PCR扩增，条件是用所述限制性内切酶消化所述线性目标双链DNA后产生两个突出的粘端，这两个粘端均是非回文结构的且不能彼此互补配对，其中对于每个酶切位点，其切割位点位于其识别序列与感兴趣的双链DNA之间，
(3)用所述限制性内切酶消化步骤(2)获得的PCR扩增产物，产生所述两端均具有非回文结构且不能彼此互补配对的粘端的线性目标双链DNA。
在一个实施方案中，可以将目标双链DNA克隆入具有两个酶切位点的质粒中，使得目标序列的两端各具有一个酶切位点，当使用识别所述酶切位点的限制性内切酶酶切质粒时可产生两端均具有非回文结构突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对。
在一个实施方案中，如下提供所述两端均具有非回文结构且不能彼此互补配对的粘端的线性目标双链DNA：
(1)提供模板质粒DNA，该模板质粒DNA中在给定序列两侧各含有至少一个酶切位点，对于每个酶切位点，其切割位点位于其识别序列与给定序列之间，使用识别所述酶切位点的限制性内切酶酶切质粒DNA时可产生一个在两端具有 非回文结构的突出的粘端的DNA片段，且这两个粘端不能彼此互补配对，优选所述酶切位点不存在于所述质粒序列的其他位置，
(2)将线性目标双链DNA克隆入所述质粒，使其两侧各具有所述至少一个酶切位点之间，
(3)用使用识别所述酶切位点的限制性内切酶消化步骤(2)获得的质粒，获得一条两端均具有非回文结构突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对。
所述两个粘端的序列可以相同或不同，优选是相同的。
在一个优选的实施方案中，上述步骤(3)获得一条具有非回文结构且彼此不互补的两个5’突出的粘端的线性目标双链DNA，更优选地，获得一条具有两个相同的非回文结构的5’突出的粘端的目标DNA。
在一个优选的实施方案中，上述步骤(3)获得一条具有非回文结构且彼此不互补的两个3’突出的粘端的线性目标双链DNA，更优选地，获得一条具有两个相同的非回文结构的3’突出的粘端的目标DNA。
如本文所用，质粒能够在生物体内复制的环状双链DNA载体，其包含复制起点、克隆位点，以及任选包含选择标记，例如抗生素抗性基因。本文所述质粒包含能够所述限制性内切酶的两个酶切位点，其中所述两个酶切位点的识别序列位于双链DNA的不同链上，由此所述内切酶切割所述质粒DNA可以获得具有彼此不互补的两个突出的粘端的目标DNA片段。
在一个实施方案中，所述质粒在同一条链上包含BsmBI酶切位点CGTCTCN 1N 2N 3N 4N 5及其互补序列，其中N 1-N 5为脱氧核糖核苷酸A、T、C、G任一，并且N 2-N 5中至少3个或所有4个选自G和C，条件是N 2N 3N 4N 5不是回文序列。
在一个实施方案中，所述质粒在同一条链上包含DNA序列CGTCTCTCGGC(SEQ ID NO：4)及其互补序列。
在一个实施方案中，所述质粒包含SEQ ID NO：1所示的核苷酸序列。
如本文所用，将目标双链DNA克隆入质粒中是指将目标DNA序列通过本领域已知的合适手段(例如使用限制性内切酶)插入在所需位置，即所述目标双链DNA的序列两端分别与所述酶切位点连接。
如本文所用，消化是在适合限制性内切酶切割DNA核酸分子的条件下，将酶与DNA分子孵育，使得限制性内切酶识别DNA分子中的限制性内切酶识别序列并相应切割DNA分子。本领域已知特定酶所需的消化条件，例如pH、温度、辅离子例如镁离子等的存在与否等。
优选地，进行连接时，在反应体系中同时存在所述限制性内切酶。这样可以有效防止被切开的部分序列重新连接回初始序列。由此，酶切后无需纯化，可以直接配制连接体系进行连接反应。
在一个方面，本发明提供了通过本发明的方法获得或可获得的线性闭合DNA。在一个实施方案中，所述线性闭合DNA的序列包含茎环部分-目标双链 DNA序列部分-茎环部分，其中所述茎环部分的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的。通过本文所述方法获得的线性闭合DNA能够抗核酸酶降解，具有提高的稳定性。
在一个方面，本发明提供了一种质粒DNA，其包含第一限制性内切酶的酶切位点、给定序列或目标序列和第二限制性内切酶的酶切位点，其中对于每个酶切位点，其切割位点位于其识别序列与给定序列或目标序列之间，其中该质粒被所述第一和第二限制性内切酶消化后能够产生一条两端均具有非回文结构突出的粘端的线性双链DNA片段，且这两个粘端不能彼此互补配对。优选地，所述质粒被所述限制性内切酶消化后产生一条具有两个5’突出的粘端或两个3’突出的粘端、特别是两个相同的5’突出的粘端或两个相同的3’突出的粘端的DNA片段，并且所述突出的粘端的序列不是回文序列。
如本文所用，给定序列是指将目标DNA克隆入质粒中时被目标DNA替换的DNA序列，其可以是任何合适的DNA序列，包括例如原核序列、多克隆位点等。
所述第一限制性内切酶和第二限制性内切酶可以相同或不同，优选是相同的。
在一个实施方案中，所述质粒在同一条链上包含DNA序列CGTCTCN 1N 2N 3N 4N 5及其互补序列，其中N 1-N 5为脱氧核糖核苷酸A、T、C、G任一，并且N 2-N 5中至少3个或所有4个选自G和C，条件是N 2N 3N 4N 5不是回文序列，优选地，N 2N 3N 4N 5为CGGC。
在一个实施方案中，所述质粒在同一条链上包含DNA序列CGTCTCTCGGC(SEQ ID NO：4)及其互补序列。
在一个实施方案中，所述质粒包含SEQ ID NO：1所示的核苷酸序列。
本发明公开的闭合线性双链DNA生产方法与现有方法相比，简单快捷且避免目的序列发生错配或突变，确保序列准确性。另外，本发明证明了通过本发明所述方法生产的闭合线性双链DNA具有表达蛋白的正常功能。因此，根据本发明的方法产生的闭合线性双链DNA可替代传统质粒DNA，具有广泛的应用前景，例如由于本发明方法简单并且生产的闭合线性GC-DNA安全性高、稳定性好，可以用于DNA疫苗领域；另外，GC-DNA也可广泛应用于基因编辑、基因治疗或细胞治疗等领域。
如本文所用，“任选”或“任选地”是指随后描述的事件或情况发生或不发生，该描述包括其中所述事件或情况发生及不发生的情况。例如，任选包括的步骤是指该步骤存在或不存在。
如本文所用，术语“约”是指包括具体数值的数值范围，本领域技术人员可以合理认为其类似于具体数值。在一些实施方案中，术语“约”是指在使用本领域通常接受的测量的标准误差内。在一些实施方案中，约是指到具体数值的+/-10％。
本文公开的范围应该认为也具体公开了所有可能的子范围以及该范围内的 各个数值。例如，对范围从1到6的描述应视为已明确公开了从1到3，从1到4，从1到5，从2到4，从2到6，从3至6等的子范围，以及该范围内的单个数字，例如1、2、3、4、5和6。无论范围的广度均适用这点。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例
实施例中涉及的主要材料包括安徽通用生物公司合成的质粒DNA(SEQ ID NO：1)及接头引物(SEQ ID NO：5)、BsmBI快速内切酶及缓冲液(美国ThermoFisher Scientific公司)、T4-DNA连接酶及缓冲液(南京诺唯赞生物技术有限公司)、Exo III核酸外切酶及缓冲液(美国ThermoFisher Scientific公司)、50×TAE缓冲液(上海生工生物工程公司)、核酸染料(上海生工生物工程公司)、琼脂糖(上海生工生物工程公司)、DNA Ladder(美国ThermoFisher Scientific公司)、293T细胞(美国ATCC中心)、聚乙烯亚胺(PEI，美国Sigma-Aldrich公司)等。
实施例1：GC-DNA产生和鉴定
1.产生线性DNA：
本实施例中使用的线性DNA是通过限制性内切酶法从模板质粒DNA中获得。所述模板质粒通过安徽通用生物公司进行基因合成和亚克隆获得，质粒上包含启动子、报告基因及polyA尾等目的序列，并且在两端添加Type IIS类限制性内切酶BsmBI的酶切位点以便将目的序列与细菌序列分隔开(图2B)。模板质粒DNA图谱见图2，核苷酸序列见SEQ IN NO：1。具体过程为：
通过无内毒素质粒大提试剂盒(北京天根生化科技有限公司)获得大量质粒DNA，按说明书配制酶切体系：将质粒DNA与BsmBI及缓冲液(美国ThermoFisher Scientific公司)混合均匀，工作温度下反应1至2小时。反应结束后，无需进行纯化，直接进行后续操作。
BsmBI酶切示意图见图3A-3C。
2.产生茎环接头：
引物序列(SEQ IN NO：5)从安徽通用生物公司合成获得。引物经过高温退火，最终形成茎环接头，且该茎环接头的末端存在可与线性目标DNA结合的粘端(图4所示)。为提高DNA的稳定性，在合成引物过程中在环区的核苷酸上加上硫代磷酸酯修饰，这种修饰可以阻止单链环的部分被核酸酶降解。将引物用无核酶水稀释后(1μM)进行退火反应，通过保持在95℃下5至10分钟来进行变性，接着缓慢降温以使茎环接头最大化形成，最终温度可降低至4℃。
3.GC-DNA形成及鉴定：
将适当包含T4-DNA连接酶(5μl/100μl反应体系)和连接缓冲液(南京诺唯赞生物技术有限公司)加入退火后的引物(即茎环接头)(20μl/100μl反应体系)和酶切后获得的线性DNA(10μg/100μl反应体系)混合物中。在16℃温度下反应16小时，或直接过夜孵育。
然后，通过乙醇沉淀法或者商品化纯化回收试剂盒对获得的连接产物进行纯化。取0.3至1μg纯化产物，加入适当Exo III核酸外切酶(美国ThermoFisher Scientific公司)及其缓冲液，混合均匀后，在37℃下反应15分钟。以正常质粒DNA为阳性对照组，及以经过酶切或PCR获得的样品为阴性对照组。反应结束后，通过凝胶电泳对样品进行检测，保持样品上样质量一致。
结果见图5，经过Exo III核酸外切酶处理后，阳性对照组中条带未消失，而阴性对照组中条带完全消失，表明Exo III核酸外切酶可用来区分闭合DNA与线性DNA。在纯化的连接产物中，经过Exo III核酸外切酶处理后，条带依然存在，表明纯化产物中存在闭合线性双链DNA。
4.线性目标DNA与茎环接头连接摩尔比：
通常，连接体系中线性目标DNA与茎环接头的摩尔比例会影响连接效率，即GC-DNA产率。本发明比较了不同摩尔比列连接产物的差异。
具体实施过程为：将线性目标DNA与茎环接头按1:1至1:15范围的不同摩尔比混合，然后加入适当T4-DNA连接酶和连接缓冲液；在16℃下反应16小时，或直接过夜孵育。反应结束后，通过乙醇沉淀法或者商品化纯化回收试剂盒对获得的连接产物进行纯化。然后取0.3至1μg纯化产物，加入适当Exo III核酸外切酶(美国ThermoFisher Scientific公司)及其缓冲液，混合均匀后，在37℃下反应15分钟。同时以正常质粒DNA为阳性对照组，及以经过酶切或PCR获得的样品为阴性对照组。反应结束后，通过凝胶电泳对样品进行检测，保持样品上样质量一致。
结果见图6，经过Exo III核酸外切酶处理后，阳性对照组中条带未消失，而阴性对照组中条带完全消失，表明反应完全。经过Exo III核酸外切酶处理后，不同比例连接产物均有条带，而1:1.5组的条带相比其他组较弱，1:3及1:6组的条带相对较强，表明线性目标DNA与茎环接头的摩尔比在1:3至1:6范围时，连接效率较强。此外，与未进行Exo III核酸外切酶处理的样品相比，1:3至1:6摩尔比范围的连接产物条带亮度变化较小，表明产物中闭合线性双链DNA(GC-DNA)比例较高。
实施例2：
1.PCR法快速制备GC-DNA：
根据模板质粒中目的片段序列设计PCR引物(SEQ IN NO：6和7)，引物中含有使用的限制性内切酶BsmBI的识别序列。使用PCR高保真酶(南京诺唯赞生物技术有限公司)，根据说明书配制PCR体系并进行PCR反应。反应完成 后，可通过凝胶电泳检测PCR产物(SEQ ID NO：8)。结果见图7A，该PCR产物除了目的条带，产物中还有2条非特异性条带，这与引物特异性有关，可以通过更换引物或者提高退火温度来减少非特异性条带的产生。
然后通过凝胶电泳或者PCR纯化试剂盒对PCR产物进行纯化回收。接着如实施例1所述酶切及连接法制备GC-DNA进行后续操作，即用限制性内切酶BsmBI对PCR产物进行酶切，并与茎环接头进行连接。连接完成后，用Exo III核酸外切酶对产物进行鉴定。结果见图7B，Exo III核酸外切酶反应后，GC-DNA中目的条带存在，表明通过PCR法可获得GC-DNA。
2.GC-DNA纯化：
为了获得更高纯度线性闭合DNA，用Exo III核酸外切酶对样品进行处理或者通过层析柱对样品进行进一步纯化。在此，对于PCR法制备的GC-DNA，由于PCR产物中存在非特异性条带，根据不同条带大小不同，利用分子筛色谱柱(苏州赛分科技股份有限公司)对体外连接产物进行纯化。
根据高效液相色谱仪(HPLC)及分子筛色谱柱说明书进行操作，流动相为Tris-HCl缓冲液(pH 7.5)。当开始出峰时，收集样品，每20秒收集一管样品，结束后可以进行浓度检测或凝胶电泳检测。结果见图8，HPLC纯化后，所用分子筛色谱柱可明显将小条带分离。样品纯化效果与色谱柱灵敏度有关，更换适合的分子筛色谱柱，有可能获得更高纯度的闭合线性双链DNA。
3.GC-DNA细胞转染及功能验证：
为了验证获得的GC-DNA是否能表达目的蛋白，将获得的GC-DNA进行细胞转染，并在荧光显微镜下观察报告基因(绿色荧光蛋白GFP(SEQ ID NO：3))是否表达。
具体实施过程为：转染前一天将293T细胞进行传代接种，细胞培养及传代方法参照《细胞培养技术》(兰容、周珍辉主编)中具体方法进行，所涉及的试剂均来源于商业化产品。当细胞汇合度达到70至80％时，将准备好的GC-DNA(1μg)通过转染导入细胞中，转染方法可以选择例如脂质体转染、钙转、PEI转染或电转。转染48小时后，可观察细胞中GFP表达。
结果见图9，闭合线性双链DNA(GC-DNA)转染后，绿色荧光蛋白可正常表达，表明生产的闭合线性双链DNA(GC-DNA)可以和传统质粒载体一样表达蛋白。
4.GC-DNA稳定性验证
为了验证GC-DNA的稳定性，本发明将获得的GC-DNA进行细胞转染，并将细胞培养7天，通过荧光定量PCR检测细胞中DNA序列的相对含量，并与转染质粒DNA及线性DNA的分组进行比较(通过测试各分组中均包含的GFP基因序列的相对含量，SEQ ID NO：3)。
具体实施过程为：转染前一天将293T细胞进行传代接种，细胞培养及传代方法参照《细胞培养技术》(兰容)中具体方法进行，所涉及的试剂均来源于商业化产品。当细胞密度达到70至80％时，将准备好的GC-DNA、线性DNA或 质粒DNA(1μg)通过转染导入细胞中，转染方法可以选择例如脂质体转染、钙转、PEI转染或电转。细胞培养3天及7天后收集细胞，细胞沉淀溶于缓冲液中保存，缓冲液可选择PBS或TE缓冲液等。收集到的细胞样品经过反复冻融3次后，离心并收集上清。根据荧光定量试剂盒说明书(诺唯赞)进行荧光定量PCR检测。
最初通过转染进入细胞的外源DNA在细胞内会逐渐被降解，也会通过细胞分裂不断被稀释。转染7天后，相同数量细胞中的外源DNA含量比转染3天后明显下降(二者相对比值小于100％)，但GC-DNA组中转染7天后的外源DNA含量相对转染3天后的百分比明显高于环状质粒组及线性DNA组(结果见图10)。这表明本发明生产的GC-DNA在细胞内的稳定性明显高于环状质粒及线性DNA。
本发明的序列
质粒DNA核苷酸序列(SEQ IN NO:1)
Figure PCTCN2022096863-appb-000001
Figure PCTCN2022096863-appb-000002
Figure PCTCN2022096863-appb-000003
绿色荧光蛋白核苷酸序列(SEQ IN NO:2)
Figure PCTCN2022096863-appb-000004
Figure PCTCN2022096863-appb-000005
绿色荧光蛋白氨基酸序列(SEQ IN NO:3)
Figure PCTCN2022096863-appb-000006
含有BsmBI酶识别位点的DNA序列(SEQ IN NO:4)
5’-CGTCTCTCGGC-3’
茎环接头引物序列(SEQ IN NO:5)
gccgATCGCGAG*A*G*G*G*T*T*G*A*CTCGCGAT(星号表示硫代修饰)PCR上游引物序列(SEQ IN NO:6)
Figure PCTCN2022096863-appb-000007
PCR下游引物序列(SEQ IN NO:7)
Figure PCTCN2022096863-appb-000008
PCR扩增片段核苷酸序列(SEQ IN NO:8)
Figure PCTCN2022096863-appb-000009
Figure PCTCN2022096863-appb-000010
Figure PCTCN2022096863-appb-000011
GFP_qPCR上游引物序列(SEQ IN NO:9)
Figure PCTCN2022096863-appb-000012
GFP_qPCR下游引物序列(SEQ IN NO:10)
Figure PCTCN2022096863-appb-000013

Claims (12)

  1. 一种制备闭合线性双链DNA的方法，包括：
    (a)提供一条含目标序列和在目标序列两端的限制性内切酶的酶切位点的双链DNA，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，其中所述限制性内切酶切割该双链DNA时可产生两端均具有非回文突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
    (b)用所述限制性内切酶消化所述双链DNA，产生一条两端均具有非回文突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
    (c)提供茎环DNA，所述茎环DNA分别具有一个与所述线性目标双链DNA两端的粘端互补配对的突出的粘端，其中，所述茎环DNA的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的，
    (d)连接(b)获得的线性目标双链DNA和(c)的茎环DNA以形成闭合线性双链DNA，优选在存在所述限制性内切酶的情况下进行连接，和
    (e)任选地，分离和/或纯化形成的闭合线性双链DNA，
    其中，除在(a)的双链DNA中酶切位点中的识别序列外，所述目标序列、所述线性目标双链DNA、所述闭合线性双链DNA均不含有所述限制性内切酶的识别序列。
  2. 权利要求1的方法，其中：
    -(a)中的双链DNA的一条链上包含：第一限制性内切酶的识别序列-第一限制性内切酶的切割位点-目标序列-第二限制性内切酶的切割位点的互补序列-第二限制性内切酶的识别序列的互补序列，优选所述第一限制性内切酶和第二限制性内切酶是相同的，和/或
    -所述线性目标双链DNA两端的粘端的序列是相同的，和/或
    -所述线性目标双链DNA具有两个5’突出的粘端或两个3’突出的粘端，优选两个相同的5’突出的粘端或两个相同的3’突出的粘端。
  3. 权利要求1或2的方法，其中步骤(a)包括：
    (i)提供目标序列，提供一对引物，该对引物分别包含限制性内切酶的酶切位点序列及目标序列的上游或下游序列，以目标序列为模板进行扩增，从而产生所述线性目标双链DNA；或者
    (iii)提供目标序列，提供质粒DNA，该质粒DNA包含给定序列和分别位于给定序列两端的至少两个酶切位点，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，将所述目标序列克隆入所述质粒的所述至少两个酶切位点之间，从而产生所述线性目标双链DNA。
  4. 权利要求1-3任一项的方法，其中使用连接酶连接所述目标DNA和茎环 DNA以形成闭合线性双链DNA，优选地，所述连接酶选自T4-DNA连接酶、T3-DNA连接酶、T7-DNA连接酶、大肠杆菌DNA连接酶、Taq DNA连接酶和热稳定5’AppDNA连接酶。
  5. 权利要求1-4任一项的方法，其包括：
    (1)提供感兴趣的线性双链DNA，
    (2)提供质粒DNA，该质粒DNA包含给定序列和分别位于给定序列两端的至少两个酶切位点，其中对于每个酶切位点，其切割位点位于其识别序列与目标序列之间，优选所述至少两个酶切位点是相同的，
    (3)将所述感兴趣的线性双链DNA克隆入所述质粒的所述至少两个酶切位点之间，其中对于每个酶切位点，其切割位点位于其识别序列与感兴趣的线性双链DNA之间，
    (4)用分别识别所述酶切位点的限制性内切酶消化所述质粒，获得一条两端均具有非回文突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
    (5)提供分别具有一个与所述线性目标双链DNA的粘端互补的突出的粘端的茎环DNA，其中，所述茎环DNA的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的，
    (6)优选在存在所述限制性内切酶的情况下，使用连接酶将步骤(4)中获得的线性目标双链DNA和步骤(5)中的茎环DNA连接以形成闭合线性双链DNA，和
    (7)任选地，分离和/或纯化形成的闭合线性双链DNA，
    优选地，其中一个酶切位点包含序列：CGTCTCN 1N 2N 3N 4N 5(SEQ ID NO：11)，其中N 1-N 5独立地为脱氧核糖核苷酸A、T、C、G任一，条件是N 2N 3N 4N 5不是回文序列，优选N 2-N 5中至少3个或所有4个选自G和C，更优选地，一个酶切位点包含序列CGTCTCTCGGC，
    更优选地，所述质粒具有SEQ ID NO：1所示的核苷酸序列。
  6. 权利要求1-4任一项的方法，其包括：
    (1)提供感兴趣的线性双链DNA，
    (2)提供一对引物，该对引物分别包含限制性内切酶的酶切位点及感兴趣的DNA序列上游序列或下游序列，以感兴趣的线性双链DNA为模板，进行扩增，产生一条含感兴趣的线性双链DNA和在感兴趣的线性双链DNA两端的限制性内切酶的酶切位点的双链DNA，其中对于每个酶切位点，其切割位点位于其识别序列与感兴趣的线性双链DNA之间，其中所述限制性内切酶切割该双链DNA时可产生两端均具有非回文突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
    (3)用所述限制性内切酶消化扩增产物双链DNA，产生一条两端均具有非回文突出的粘端的线性目标双链DNA，且这两个粘端不能彼此互补配对，
    (4)提供分别具有一个与所述线性目标双链DNA的粘端互补的突出的粘端的茎环DNA，其中，所述茎环DNA的环状部分中的一或多个核苷酸是修饰的，例如被硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰，优选是硫代磷酸酯修饰的，
    (5)优选在存在所述限制性内切酶的情况下，使用连接酶将步骤(3)中获得的线性目标双链DNA和步骤(4)中的茎环DNA连接以形成闭合线性双链DNA，和
    (6)任选地，分离和/或纯化形成的闭合线性双链DNA。
  7. 权利要求1-6任一项的方法，其中所述突出的粘端的长度为4-10个核苷酸，优选为4-6个核苷酸，更优选为4个核苷酸，和/或所述突出的粘端主要由G和C组成，优选G+C含量占突出的粘端中总核苷酸含量的50％以上，更优选所述突出的粘端仅由G和C组成，例如所述突出的粘端序列为CGGC。
  8. 权利要求1-7任一项的方法，其中所述限制性内切酶选自能产生非回文结构粘端的酶，例如选自BsmBI，BaeI，BbsI，BspQI，FspEI和I-CeuI，
    优选地，所述限制性内切酶是BsmBI并且所述限制性内切酶的酶切位点包含CGTCTCN 1N 2N 3N 4N 5，其中N 1-N 5独立地为脱氧核糖核苷酸A、T、C、G任一并且N 2-N 5中至少3个或所有4个选自G和C，条件是N 2N 3N 4N 5不是回文序列，更优选地，所述酶切位点为CGTCTCTCGGC(SEQ ID NO：4)。
  9. 权利要求1-8任一项的方法，其中所述茎环DNA的长度为个20-50个核苷酸，优选为20-40个核苷酸，更优选为20-30个核苷酸，和/或所述茎环DNA的环状部分长度为5-20个核苷酸，优选为5-15个核苷酸，更优选为10-15个核苷酸，
    更优选地，所述茎环DNA包含序列：
    GCCGATCGCGAG*A*G*G*G*T*T*G*A*CTCGCGAT(SEQ ID NO：5)，其中星号表示的核苷酸中的一或多个、优选全部是被修饰的，例如是硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基或醛基修饰的，优选是硫代磷酸酯修饰的。
  10. 权利要求1-9任一项的方法，其中，在连接时，所述目标DNA和茎环DNA的摩尔比例为1:1至1:15，优选为1:3至1:6。
  11. 通过权利要求1-10任一项的方法获得或可获得的线性闭合双链DNA，其中所述线性闭合双链DNA的环状部分的一或多个核苷酸是修饰的，例如是硫代磷酸酯、羧基、氨基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二 硫基或醛基修饰的，优选是硫代磷酸酯修饰的。
  12. 一种质粒，其包含给定序列或目标序列和分别在给定序列或目标序列两端的至少两个限制性内切酶的酶切位点，其中对于每个酶切位点，其切割位点位于其识别序列与给定序列或目标序列之间，其中所述限制性内切酶在切割该质粒时，产生一条两端均具有非回文结构突出的粘端的线性双链目标DNA片段，且这两个粘端不能彼此互补配对，
    优选地，所述质粒在一条链上包含：第一限制性内切酶的识别序列-第一限制性内切酶的切割位点-目标序列-第二限制性内切酶的切割位点的互补序列-第二限制性内切酶的识别序列的互补序列，更优选地，所述第一限制性内切酶和第二限制性内切酶是相同的，
    优选地，所述质粒被所述限制性内切酶消化后产生一条具有两个5’突出的粘端或两个3’突出的粘端、更优选两个相同的5’突出的粘端或两个相同的3’突出的粘端的线性双链目标DNA片段，
    优选地，所述质粒包含序列CGTCTCN 1N 2N 3N 4N 5及其互补序列，其中N 1-N 5独立地为脱氧核糖核苷酸A、T、C、G任一并且N 2-N 5中至少3个或所有4个选自G和C，条件是N 2N 3N 4N 5不是回文序列，更优选地，N 2N 3N 4N 5为CGGC，更优选地，所述质粒包含序列CGTCTCTCGGC(SEQ ID NO：4)，更优选地，所述质粒包含SEQ ID NO：1所示的核苷酸序列。
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