阅读说明：本技术 一种长单链dna的制备方法 (Preparation method of long single-strand DNA ) 是由 顾宏周 张俏 夏凯 于 2019-10-17 设计创作，主要内容包括：本发明公开了一种借助I类和II类水解性脱氧核酶高效制备长单链DNA的方法。该方法主要包括设计并构建重组噬菌粒,获得噬菌粒环状单链DNA步骤,同时采用I类脱氧核酶和II类脱氧核酶突变体切割环状单链DNA,纯化回收酶切得到的所述长单链DNA。利用这两类能快速水解DNA的脱氧核酶,可代替限制性内切酶,低成本的实现对辅助噬菌体法制备出的DNA序列的特异性切割,大量、经济、高纯度制备任意长度和序列的单链DNA。(The invention discloses a method for efficiently preparing long single-stranded DNA by means of class I and class II hydrolytic deoxyribozymes. The method mainly comprises the steps of designing and constructing recombinant phagemid to obtain phagemid circular single-stranded DNA, simultaneously adopting I-type deoxyribozyme and II-type deoxyribozyme mutant to cut the circular single-stranded DNA, and purifying and recovering the long single-stranded DNA obtained by enzyme digestion. The two types of deoxyribozymes capable of rapidly hydrolyzing DNA can replace restriction endonucleases, realize the specific cutting of the DNA sequence prepared by the helper phage method at low cost, and prepare single-stranded DNA with any length and sequence in large quantity, economy and high purity.)

1. A method for preparing long single-stranded DNA, comprising the step of cleaving circular single-stranded DNA by using both a class I deoxyribozyme and a class II deoxyribozyme mutant.

2. The method according to claim 1, wherein the group I deoxyribozyme is I-R3, and the substrate domain sequence thereof is shown in SEQ ID NO: 1, the enzyme domain sequence is shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the mutant DNAzyme II is one of II-R1a, II-R1b, II-R1c and II-R1 d.

4. The production method according to claim 3,

the substrate domain sequence of the II-R1a deoxyribozyme mutant is shown as SEQ ID NO: 3, the enzyme domain sequence is shown as SEQID NO: 4; or

The substrate domain sequence of the II-R1b deoxyribozyme mutant is shown as SEQ ID NO: 5, the enzyme domain sequence is shown as SEQ ID NO: 6; or

The substrate domain sequence of the II-R1c deoxyribozyme mutant is shown as SEQ ID NO: 7, the enzyme domain sequence is shown as SEQ ID NO: 8; or

The substrate domain sequence of the II-R1d deoxyribozyme mutant is shown as SEQ ID NO: 9, the enzyme domain sequence is shown as SEQ ID NO: 10, or a fragment thereof.

5. The method of claim 3or 4, wherein the step of cleaving the circular single-stranded DNA is preceded by a step of designing and constructing a recombinant phagemid and a step of obtaining a phagemid circular single-stranded DNA.

6. The method of claim 5, wherein the designing and constructing of the recombinant phagemid comprises adding the substrate domain sequences of the class I deoxyribozyme and the class II deoxyribozyme mutant, or the class I deoxyribozyme and the class II deoxyribozyme mutant, respectively, to the 5 'end and the 3' end of the target single-stranded DNA sequence.

7. The preparation method of claim 5, wherein the obtaining of the phage circular single-stranded DNA comprises transforming the constructed recombinant phagemid into Escherichia coli cells for replication, then infecting with helper phage, centrifuging to remove Escherichia coli, collecting supernatant and precipitating phage particles, and removing phage protein coat by alkaline lysis to obtain the circular single-stranded DNA.

8. The method according to claim 1, further comprising, after the step of cleaving the circular single-stranded DNA, purifying and recovering the long single-stranded DNA obtained by the cleavage.

9. The method according to claim 1, wherein the temperature for cleaving the circular single-stranded DNA is 37 ℃ to 50 ℃ for 0.5h to 24 h.

10. Use of the long single-stranded DNA prepared by the method of any one of claims 1 to 9 or the method of any one of claims 1 to 9 in DNA nano-technology, gene editing, gene therapy, DNA probes, and the like.

Technical Field

The invention belongs to the fields of biochemistry and molecular biology, and particularly relates to a method for preparing long single-stranded DNA by combining two types of deoxyribozymes capable of rapidly hydrolyzing DNA with helper phage.

Background

Currently, DNA nanotechnology, the field of biomedical research such as knock-in, has a wide demand for Single strand DNA (ssDNA), especially for long Single strand DNA (>100 bases). However, due to the limitation of chemical synthesis methods, the synthesis of long single-stranded DNA is difficult to ensure yield, yield and satisfactory cost performance, so that the in vivo or in vitro action of biological enzymes and some auxiliary denaturation means are required for the long single-stranded DNA, and the currently commonly used preparation methods of the long single-stranded DNA mainly include a reverse transcription method, an enzyme degradation method, a denaturation High Performance Liquid Chromatography (HPLC) method, a Biotin-on-beads (Biotin) modification method, an asymmetric PCR method, an RCA method and the like. However, in practical applications, these methods all have problems of low yield, high cost, and the like.

The helper phage method is a relatively new method for preparing single-stranded DNA. The basic principle is to construct a plasmid containing either the M13 origin of replication (M13ori) or the F1 origin of replication (F1ori), transfer it into a host cell containing the F' factor, and then infect it with a defective helper phage. Helper phage can help the plasmid form single-stranded DNA and pack into phage, secreting out of the host cell (as shown in FIG. 1). The method has low cost and high yield, and is very suitable for preparing long single-stranded DNA sequences. However, the resulting single-stranded DNA is circular and contains an essential conserved M13ori/f1ori sequence. If the traditional restriction enzyme digestion method is adopted to obtain the desired single-stranded DNA part, the preparation cost is greatly increased. At the same time, the method is limited by the dependence of the endonuclease on the DNA recognition sequence.

Deoxyribozymes (deoxyribozymes) are single-stranded DNA fragments with catalytic function, have high catalytic activity and structure recognition capability, and can catalyze a plurality of chemical reactions including DNA phosphorylation, adenylation, deglycosylation and the like. In recent years, Zn has been screened by some researchers²⁺As a cofactor, a deoxyribozyme capable of hydrolyzing a phosphodiester bond of DNA at a specific site, and hydrolyzing a single-stranded DNA using the deoxyribozyme. However, in the prior art, the class I deoxyribozyme is used for a cleavage reaction, two bases AG are left at the 5 'end of the obtained single-stranded DNA, and five bases GTTGA are left at the 3' end, so that the complete self-definition of a single-stranded DNA sequence cannot be realized, and the requirements in practical applications such as probe preparation cannot be completely met.

Disclosure of Invention

In order to solve the technical problems, the invention uses two types of deoxyribozymes capable of quickly hydrolyzing DNA to replace restriction endonucleases, can realize the specific cutting of a DNA sequence prepared by a helper phage method at low cost, and obtains single-stranded DNA with any length and sequence.

In one aspect, the present invention provides a method for preparing long single-stranded DNA, comprising the step of cleaving circular single-stranded DNA using both a class I dnazyme and a class II dnazyme mutant.

Alternatively, the I type deoxyribozyme is I-R3, and the sequence of the substrate domain of the I type deoxyribozyme is shown in SEQ ID NO: 1, the enzyme domain sequence is shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

Alternatively, the mutant of the group II deoxyribozyme is one of II-R1a, II-R1b, II-R1c and II-R1 d. The substrate domain sequence of the II-R1a deoxyribozyme mutant is shown as SEQ ID NO: 3, the enzyme domain sequence is shown as SEQ ID NO: 4; the substrate domain sequence of the II-R1b deoxyribozyme mutant is shown as SEQ ID NO: 5, the enzyme domain sequence is shown as SEQ ID NO: 6; the substrate domain sequence of the II-R1c deoxyribozyme mutant is shown as SEQ ID NO: 7, the enzyme domain sequence is shown as SEQ ID NO: 8; the substrate domain sequence of the II-R1d deoxyribozyme mutant is shown as SEQ ID NO: 9, the enzyme domain sequence is shown as SEQ ID NO: 10, or a fragment thereof. The stem region sequence of the II-type deoxyribozyme mutant is any nucleotide sequence.

Alternatively, in the step of cleaving the circular single-stranded DNA, DNAzyme cleavage reaction buffer 1(50mM HEPES, 100mM LiCl, pH7.0) is added to the collected circular single-stranded DNA.

If the deoxyribozyme is in a two-chain form, namely split into a substrate chain and a enzyme chain for cutting, the substrate domain sequences of the I type deoxyribozyme and the II type deoxyribozyme mutant are respectively added at two ends of a target sequence through PCR, and the corresponding deoxyribozyme enzyme sequence is additionally added when the deoxyribozyme is subjected to hydrolytic cutting. Wherein, the corresponding II type deoxyribozyme mutant is selected according to the last base at the 3' end of the single-chain sequence to be prepared. If the last base at the 3' end is G, II-R1a is selected; if the last base at the 3' end is A, II-R1b is selected; if the last base at the 3' end is T, II-R1c is selected; if the last base at the 3' end is C, II-R1d is selected.

If the deoxyribozyme substrate domain and the enzyme domain are on one sequence and cut in the form of one sequence, I-type deoxyribozyme and II-type deoxyribozyme mutant sequences are added at two ends of the target sequence respectively through PCR.

After denaturation annealing, DNAzyme cleavage reaction buffer 2(50mM HEPES, 100mM LiCl, 20mM MgCl) was added₂，4mM ZnCl₂pH7.0) is adopted, the cutting is carried out in the range of 37 ℃ to 50 ℃, the specific reaction temperature is determined according to the length of a stem region of the deoxyribozyme, and the cutting time is different from half an hour to 24 hours according to actual requirements.

Optionally, before the step of cutting the circular single-stranded DNA, the steps of designing and constructing a recombinant phagemid and obtaining the phagemid circular single-stranded DNA are also included.

Alternatively, the recombinant phagemid can be designed and constructed to have the length and sequence of the target single-stranded DNA according to different application requirements. Different plasmids or different biological genomes can be used as templates for PCR amplification to obtain DNA fragments, or the DNA fragments can be directly chemically synthesized; the length of the DNA fragment can be designed to vary from several tens of base pairs to several tens of thousands of base pairs.

Alternatively, the designing and constructing of the recombinant phagemid comprises adding a class I deoxyribozyme and a class II deoxyribozyme mutant sequence, or adding a class I deoxyribozyme and a class II deoxyribozyme mutant substrate domain sequence, respectively, to the 5 'end and the 3' end of the target single-stranded DNA sequence. On the basis, enzyme cutting sites or vector homologous sequences are added on both sides of the DNA fragment. The amplified DNA fragment can be ligated to a phagemid vector containing M13ori or f1ori by digestion, ligation or homologous recombination to construct a recombinant phagemid.

Alternatively, the obtaining of phage circular single-stranded DNA comprises transforming the recombinant phagemid constructed as described above into E.coli cells (e.g., JM109, XL-1blue) containing factor F, and replicating it in large amounts in E.coli. The recombinant phagemid will then be packaged in single stranded form in the phage by infection with a helper phage (e.g. M13KO7, VCSM13) and secreted into the cell culture broth. Escherichia coli is removed by centrifugation, the supernatant is collected and phage particles are precipitated, and then the shell of phage protein is stripped by an alkaline lysis method, so that the corresponding circular single-stranded DNA can be obtained.

Optionally, after the step of cleaving the circular single-stranded DNA, further comprising purifying and recovering the long single-stranded DNA obtained by the cleavage, specifically, after the cleavage reaction is completed, selecting an agarose gel or a polyacrylamide gel with a suitable concentration for purification according to the length of the target single-stranded DNA sequence, and removing the redundant vector sequence and the deoxyribozyme sequence. The target single-stranded DNA can be recovered by a gel recovery kit or a gel elution buffer solution.

In a second aspect, the invention also provides the application of the preparation method of the long single-stranded DNA in DNA nano-grade, gene editing, gene therapy and the like.

In a third aspect, the invention also provides the application of the long single-stranded DNA prepared by the preparation method of the long single-stranded DNA in DNA nano-grade, gene editing, gene therapy, DNA probes and the like. Alternatively, in knock in experiments.

Compared with the prior art, the invention has the beneficial effects that:

1) in the prior art, class I deoxyribozymes are used for carrying out a cleavage reaction, two AG basic groups are left at the 5 'end of the obtained single-stranded DNA, and five GTTGA basic groups are left at the 3' end of the single-stranded DNA, but the invention utilizes the class I deoxyribozymes and the class II deoxyribozymes mutants to carry out the cleavage reaction simultaneously, so that the prepared single-stranded DNA only has two AG basic groups at the 5 'end, no residual basic groups at the 3' end, and the cleavage efficiency can reach more than 70%. In practical application, the residual AG base at the 5' end can be designed to be included in the single-stranded DNA sequence to be prepared, thereby realizing the complete self-definition of the single-stranded DNA sequence. Compared with the prior art, the invention can better meet the requirements of applications (such as DNA probes and the like) which have requirements on target single-chain two-end base sequences.

2) The method simultaneously utilizes two types of deoxyribozymes capable of quickly hydrolyzing DNA, can replace restriction endonucleases, and realizes the specific cutting of the DNA sequence prepared by the helper phage method at low cost.

3) The method can cut the circular DNA into DNA single strands with customized lengths according to different experimental requirements, and has high cutting effect and high purity of cut fragments.

4) The invention provides a II-type hydrolytic deoxyribozyme mutant capable of efficiently preparing long single-stranded DNA, and the cleavage rate can be rapidly improved.

Drawings

FIG. 1 is a schematic diagram of the preparation of single-stranded DNA by the helper phage method in the prior art;

FIG. 2 is a schematic diagram showing the cleavage of single-stranded DNA by a deoxyribozyme according to the present invention;

FIG. 3 is a comparison graph of the advantages of the single-stranded DNA prepared by the present invention and the prior art;

FIG. 4 is an electrophoretogram of single-stranded DNAs of different sequences and sizes prepared according to the present invention, the single-stranded DNAs being 1500nt and 517nt in length, respectively;

FIG. 5 is the electrophoresis chart of single-stranded DNA with different sequences and sizes prepared by the present invention, wherein the length of the single-stranded DNA is 160nt and 60nt respectively; meanwhile, FIG. 5(a) and FIG. 5(b) show the comparison results of the purity of single-stranded DNA in example 2, respectively;

FIG. 6 is a schematic view of knock in principle;

FIG. 7 is a graph showing the results of the cytotoxicity test in example 3;

FIG. 8 is a graph of the results of confocal laser microscopy imaging after transfection of cells in example 3.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby. It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.