WO2023115786A1 - 一种制备双链rna的方法 - Google Patents

一种制备双链rna的方法 Download PDF

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WO2023115786A1
WO2023115786A1 PCT/CN2022/092023 CN2022092023W WO2023115786A1 WO 2023115786 A1 WO2023115786 A1 WO 2023115786A1 CN 2022092023 W CN2022092023 W CN 2022092023W WO 2023115786 A1 WO2023115786 A1 WO 2023115786A1
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double
stranded
stranded dna
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circular
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梁兴国
陈辉
安然
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中国海洋大学
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

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  • the invention belongs to the technical field of nucleic acid, and in particular relates to a method for preparing double-stranded RNA.
  • Double-stranded RNA (dsRNA) structure is ubiquitous in nature, and it is often used as a small part of long-chain RNA, such as ribosomal RNA (rRNA), messenger RNA (mRNA) and viroid (a virus composed only of RNA). part.
  • rRNA ribosomal RNA
  • mRNA messenger RNA
  • viroid a virus composed only of RNA. part.
  • RNA interference RNA interference
  • siRNAs small interfering RNAs
  • RNAi technology has been tried to be applied in agriculture (insect pest control) (Insect Sci, 2013, 20, 4-14; Pest Manag Sci, 2016, 72, 801-809) and aquaculture (shrimp disease control) (J Biotechnol, 2020, 321, 48-56; J Virol Methods, 2013, 188, 64-69; IOP Conf Ser Earth Environ Sci, 2020, 584, 012051.).
  • insect pest control Insect pest control
  • aquaculture shrimp disease control
  • J Biotechnol, 2020, 321, 48-56 J Virol Methods, 2013, 188, 64-69
  • dsRNA can be prepared in vivo/in vitro (Anal Bioanal Chem, 2018, 410, 3239-3252).
  • E.coli Escherichia coli
  • plant cells UK Patent, No.WO2020183416-A1, Sep17, 2020
  • mammalian cells Methodhods Mol Biol, 2013,942,291-314.
  • enzymatic method is easier to achieve large-scale production because it is carried out in a liquid phase environment, and since no harmful organic reagents are involved in the whole process, the product safety is better (Isr J Chem,2013 , 53, 326-349; Chimia, 2005, 59, 812-816).
  • enzymatic methods can satisfy the synthesis of modified RNA and have good development prospects (Org Biomol Chem, 2018, 16, 5800-5807; Molecules, 2020, 25, 5492).
  • the existing dsRNA enzymatic synthesis methods can be divided into linear transcription and rolling circle transcription.
  • Linear transcription can be divided into two cases, one is to use two linear double-stranded DNA templates to transcribe the sense strand and antisense strand respectively, and then anneal and hybridize to obtain dsRNA (Nucleic Acids Res, 2002, 30, e46; Nucleic Acids Res, 2003 ,31,e38); the other is to directly use a linear double-stranded DNA template containing a palindromic sequence to prepare a hairpin-shaped RNA containing a sense sequence and an antisense sequence, and then process it with Dicer enzyme in vivo to obtain the target siRNA ( Mol Biotechnol, 2017, 59, 73-83).
  • the linear double-stranded DNA templates used in the above two methods all contain promoter sequences, and in order to initiate transcription efficiently, the first two bases downstream of the promoter in some templates must be GG. To obtain accurate products, Subsequent enzyme digestion is required to remove the redundantly transcribed CC (Nucleic Acids Res, 2003, 31, e38). It can be seen that the above-mentioned linear transcription methods are cumbersome and inefficient, making it difficult to meet the needs of practical applications. Rolling circle transcription is much more efficient than linear transcription. The existing rolling circle transcription methods can also be divided into two categories.
  • the present invention provides a method for preparing double-stranded RNA.
  • double-stranded RNA first design a method containing 5 - 15bp bubble structure (Bubble), circular double-stranded DNA template without promoter; then add the above-mentioned circular double-stranded DNA template, RNA polymerase, ribonuclease H, NTPs, RNase inhibitor into the system at the same time , Inorganic pyrophosphatase for transcription.
  • Bubble bubble structure
  • RNA polymerase performs non-biased high-efficiency bi-directional rolling circle transcription, and RNase H, with the assistance of the unhybridized DNA portion at the bubble structure in the template, realizes the enzyme digestion of the transcript in time.
  • RNase H ribonuclease H and circular double-stranded DNA template containing bubble structure without promoter, the one-step, efficient and mass preparation of target double-stranded RNA product is realized.
  • the method is very simple and efficient, has low cost and strong operability, and is suitable for large-scale industrial production.
  • a method for preparing double-stranded RNA comprising the steps of:
  • Step 1 designing and preparing a circular double-stranded DNA template, the circular double-stranded DNA template contains a non-complementary vesicle structure part and a complementary part without a promoter sequence, and the complementary part is compatible with the double-stranded RNA to be prepared
  • T in the sequence corresponds to U
  • the bubble structure is located at the two ends of the complementary part of the circular double-stranded DNA template, the length of the bubble structure is 5-15bp, between the two strands of the bubble structure No more than 2 consecutive complementary bases;
  • Step 2 purifying the prepared circular double-stranded DNA template to remove linear single-stranded DNA mixed in the circular double-stranded DNA template;
  • Step 3 mixing the purified circular double-stranded DNA template, RNA polymerase, ribonuclease H, NTPs, RNase inhibitors, inorganic pyrophosphatase and corresponding RNA polymerase buffer for transcription, the RNA polymerase
  • the enzyme is T7 RNA polymerase, SP6 RNA polymerase or T3 RNA polymerase.
  • RNA polymerase performs high-efficiency bidirectional rolling circle transcription without preference, and ribonuclease H is bubbly in the circular double-stranded DNA template.
  • the digestion of transcription products containing repeated sequences can be realized in time, and the target double-stranded RNA monomer that meets the requirements can be obtained.
  • sequences of the upper and lower strands of the vesicle structure may be the same or different.
  • the formation of hairpin structures or polyA, polyT and other sequences should be avoided in the upper and lower strands of the bubble structure.
  • the size of the vesicles should be between 5-15 bases, otherwise the yield of double-stranded RNA will be reduced.
  • the length of the double-stranded RNA product monomer that can be prepared by the present invention is more than 70bp; preferably 70-1000bp, more preferably 90-437bp, more preferably 96- 278bp.
  • a one-step method is used to design a circular precursor linear strand to prepare circular single-stranded DNA, and then add the circular single-stranded DNA and linear single-stranded DNA after annealing and hybridization DNA ligase and corresponding ligase buffer for ligation reaction;
  • the circular single-stranded DNA is prepared by a one-pot method or a step-by-step method, and then the circular single-stranded DNA is mixed with a plurality of short DNA strands corresponding to its complementary strand, After annealing and hybridization, add DNA ligase and corresponding ligase buffer for ligation reaction;
  • the distance between the two ends of the linear single-stranded DNA and the edge of the bubble structure should be greater than 15 nt.
  • the circular single-stranded DNA is prepared by one-step circularization; when the circular double-stranded DNA
  • design multiple segments of 40-90nt Phosphorylated DNA fragments and corresponding splints are used in one-pot, one-step or step-by-step ligation reactions to prepare circular single-stranded DNA or linear single-stranded DNA.
  • the annealing and hybridization conditions are as follows: firstly keep warm at 90° C. for 1-3 minutes, then lower the temperature to 20-25° C. at a rate of 0.1° C./s and keep warm for 10 minutes.
  • T4DNA ligase and corresponding T4DNA ligase buffer incubate at 4-37°C for 2-24h for ligation; when using TaqDNA ligase and corresponding TaqDNA ligase buffer, at 45- Insulate at 80°C for 2-24h for connection.
  • a buffer solution with a final concentration of 0.05-1 ⁇ is used.
  • the buffer solution concentration is 1 ⁇ , its composition is: 40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 500 ⁇ M ATP ( pH 7.8@25°C);
  • a buffer with a final concentration of 1 ⁇ which consists of: 20mM Tris-HCl, 25mM KAc, 10mM Mg(Ac) 2 , 10mM DTT, 1.0mM NAD , 0.1% Triton X-100 (pH 7.6@25°C).
  • Exo I and Exo III are used to digest together to remove all the linear single-stranded DNA mixed in the circular double-stranded DNA template, and the digestion of Exo I
  • the reaction uses a buffer with a final concentration of 0.5 ⁇ , and its composition is: 33.5mM glycine-KOH (pH 9.5@25°C), 3.35mM MgCl 2 , 0.5mM DTT; the enzyme digestion reaction of Exo III uses a final concentration of 0.5 ⁇ Buffer, the composition of which is: 33mM Tris-HCl (pH 8.0@30°C), 0.33mM MgCl 2 .
  • step 3 double-stranded RNA is prepared by constant temperature reaction at 37°C for 0.5-72h.
  • RNA polymerase uses a buffer solution with a final concentration of 1 ⁇ , and its composition is: 40mM Tris-HCl (pH 7.9@25°C), 6.0mM MgCl 2 , 10mM DTT , 10 mM NaCl and 2 mM spermidine.
  • the transcription template used in the method for preparing double-stranded RNA of the present invention does not need to introduce a promoter sequence, so the redundant sequence generated by the transcription of the promoter will not be introduced into the transcription product, which greatly facilitates the subsequent product purification process.
  • the two-way transcription efficiency is the same, which avoids the occurrence of an excess of one RNA strand caused by asymmetric transcription, and is especially suitable for the preparation of double-stranded RNA.
  • the method for preparing double-stranded RNA involved in the present invention is very simple, easy to operate, does not require harsh conditions, only needs to expand the reaction system, prolong the transcription time, and supply enough NTPs for the transcription system
  • the mass preparation of the target double-stranded RNA product can be realized.
  • the double-stranded RNA prepared by this method has a high purity (only 2-8 nt at both ends of the head and tail are single-stranded RNA). If the purity of the product is high, RNase A/RNase 1/RNase T1 and other enzymes can be used to remove the head and tail. For the single-stranded RNA at both ends, use DNase I and other DNases to remove the circular double-stranded DNA template, and further purify the product by extraction, alcohol precipitation, and HPLC.
  • the present invention greatly simplifies the preparation process of double-stranded RNA and has good application potential.
  • Fig. 1 embodiment 1 prepares the schematic diagram of double-stranded RNA
  • Example 1 uses B15 to prepare the results of double-stranded RNA (prepared with T7 RNA polymerase), wherein A is a schematic diagram of the sequence design of B15, and B is a schematic diagram of the results prepared by B15 at 75°C; C is a schematic diagram of the preparation of double-stranded RNA with B15 The results of RNA and the results of enzyme digestion verification of transcripts;
  • Example 2 results of double-stranded RNA preparation with B12, wherein A is a schematic diagram of the sequence design of B12, B is a schematic diagram of the results prepared by B12 at 75°C; C is the result of preparation of double-stranded RNA with B12 and the transcriptional product Enzyme digestion verification results;
  • Example 3 The results of double-stranded RNA preparation with B9, wherein A is a schematic diagram of the sequence design of B9, B is a schematic diagram of the results prepared by B9 at 75°C; C is the result of double-stranded RNA preparation with B9 and the transcripts Enzyme digestion verification results;
  • FIG. 7 Example 4 using B5 to prepare double-stranded RNA results, wherein A is a schematic diagram of the sequence design of B5, B is a schematic diagram of the results prepared by B5 at 75°C; C is the result of using B5 to prepare double-stranded RNA and the transcripts Enzyme digestion verification results;
  • FIG. 8 Example 5 using B9T to prepare double-stranded RNA results, wherein A is a schematic diagram of the sequence design of B9T, B is a schematic diagram of the results prepared by B9T at 75°C; C is the result of using B9T to prepare double-stranded RNA and the transcription product Enzyme digestion verification results;
  • FIG. 9 Example 6 using B5T to prepare the results of double-stranded RNA, wherein A is a schematic diagram of the sequence design of B5T, B is a schematic diagram of the results prepared by B5T at 75°C; C is the result of using B5T to prepare double-stranded RNA and the transcripts Enzyme digestion verification results;
  • Example 7 The results of double-stranded RNA preparation using loop 96, wherein A is a schematic diagram of the sequence design of loop 96, B is a schematic diagram of the results of loop 96 prepared at 65 ° C; C is the result of using loop 96 to prepare double-stranded RNA and Enzyme digestion verification results of transcripts;
  • Example 8 The results of double-stranded RNA preparation with loop 155, wherein A is a schematic diagram of the sequence design of loop 155, B is a schematic diagram of the results of loop 155 prepared at 65°C; C is the result of using loop 155 to prepare double-stranded RNA and Enzyme digestion verification results of transcripts;
  • FIG. 12 Example 9 The result of using loop 278 to prepare double-stranded RNA, wherein A is a schematic diagram of the sequence design of loop 278, B is a schematic diagram of the result of loop 278 prepared at 65°C; C is the result of using loop 278 to prepare double-stranded RNA and Enzyme digestion verification results of transcripts;
  • Example 10 uses B15 to prepare the result of double-stranded RNA (prepared with SP6 RNA polymerase).
  • the circular precursor linear strands used in the following examples for the preparation of circular double-stranded DNA templates were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. and were artificially synthesized; ribonuclease H (RNase H), 10 ⁇ RNase H buffer, Taq DNA ligase (Taq Dnl), 10 ⁇ Taq Dnl buffer, inorganic pyrophosphatase (Inorganic Pyrophosphatase, E.coli), ShortCut RNase III and its corresponding 10 ⁇ buffer were purchased from Anolon (Beijing) Biotechnology Co., Ltd.
  • T7 RNA polymerase T7 RNAP
  • SP6 RNA polymerase SP6 RNA polymerase
  • 5 ⁇ RNAP buffer nuclease inhibitor (RiboLock RNase Inhibitor)
  • T4 polyphosphate kinase T4 PNK
  • 10 ⁇ T4 PNK buffer A ATP (concentration of stock solution is 100mM)
  • T4 DNA ligase T4 Dnl
  • 10 ⁇ T4 Dnl buffer Exo I, 10 ⁇ buffer corresponding to Exo I, Exo III, corresponding to Exo III
  • the 10 ⁇ buffer, DNase I, 10 ⁇ DNase I buffer (containing Mg 2+ ), NTPs (collectively referred to as ATP, UTP, CTP and GTP, the stock solution concentration is 100mM) were purchased from American Thermo Fisher Biotechnology Co., Ltd. ; Nucleic acid dye solution (Ultra GelRed) was purchased from Novozyme (Nanjing) Biotechnology Co.,
  • ACCGGAGTCTGCACAA length is 16nt, SEQ ID NO: 4;
  • GTGCAGACTCCGGTGG (length is 16nt, SEQ ID NO: 7);
  • CTTTATAAACAACCAG (16 nt in length, SEQ ID NO: 8).
  • the cyclic precursor linear chain (C1-a and C1-b, final concentration of 1 ⁇ M), splint (Splint C1-ab and Splint C1-ba, final concentration of 2 ⁇ M), T4 DNA ligase (T4 Dnl, final concentration of 0.125U/ ⁇ L) and the corresponding T4 DNA ligase buffer (final concentration: 1 ⁇ ), react at 37°C for 12h to prepare C1.
  • composition of 1 ⁇ T4 Dnl Buffer 40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 500 ⁇ M ATP (pH 7.8@25°C).
  • Precursor linear strands (A-a and A-b, final concentration of 1 ⁇ M), splint (Splint A-ab, final concentration of 2 ⁇ M), T4 DNA ligase (T4 Dnl, final concentration of 0.125 U/ ⁇ L) and corresponding T4 DNA Ligase buffer (final concentration: 1 ⁇ ) was mixed and reacted at 37° C. for 12 hours to prepare LA.
  • composition of 1 ⁇ T4 Dnl Buffer 40mM Tris-HCl, 10mM MgCl 2 , 10mM DTT, 500 ⁇ M ATP (pH 7.8@25°C)
  • composition of 1 ⁇ Taq Dnl Buffer 20mM Tris-HCl, 25mM KAc, 10mM Mg(Ac) 2 , 10mM DTT, 1mM NAD, 0.1% Triton X-100 (pH 7.6@25°C).
  • Exo I final concentration about 1U/ ⁇ L
  • Exo III final concentration about 4U/ ⁇ L
  • their corresponding buffers each final concentration is 0.5 ⁇
  • composition of 0.5 ⁇ Exo I buffer is: 33.5mM glycine-KOH (pH 9.5@25°C), 3.35mM MgCl 2 , 0.5mM DTT;
  • composition of 0.5 ⁇ Exo III buffer is: 33mM Tris-HCl (pH 8.0@30°C), 0.33mM MgCl 2 .
  • composition of 1 ⁇ RNAP buffer is: 40mM Tris-HCl (pH 7.9@25°C), 6mM MgCl 2 , 10mM DTT, 10mM NaCl and 2mM spermidine.
  • ShortCut RNase III final concentration: 0.2U/ ⁇ L
  • ShortCut Reaction buffer final concentration: 1 ⁇
  • MnCl 2 final concentration: 1 ⁇
  • the experimental results were detected by 8% urea denatured polyacrylamide gel electrophoresis, and the results of the preparation of the circular double-stranded DNA template, the corresponding double-stranded RNA preparation (transcription results) and the results of ShortCut RNase III were analyzed by Image Lab software. Enzyme digestion results of transcripts.
  • Figure 4 is the result map, in which A in Figure 4 is a schematic diagram of the sequence design of B15, which contains a 15bp bubble structure with the same upper and lower chain sequences and opposite directions; B in Figure 4 is a schematic diagram of the result of B15 prepared at 75°C ; C in Figure 4 is the result of preparing double-stranded RNA with B15 (only T7 RNAP or T7 RNAP and RNase H coexist in the system) and the result of the enzymatic digestion verification of the transcript by ShortCut RNase III, and the label "S" in the figure refers to Results of ShortCut RNase III digestion.
  • the transcripts were poly-tandem repeats blocked at the gel pore; when T7 RNAP and RNase H coexisted in the transcription system, the transcripts were concentrated in a fixed length; ShortCut After RNase III enzyme digestion, the above-mentioned transcripts are all shortened, and the length is concentrated in a small fixed region. According to the enzyme's cutting characteristics (it can cut long double-stranded RNA enzyme into short double-stranded RNA of 18-25bp) , it can be determined that the transcript of B15 is the double-stranded RNA we want.
  • ACCGGAGTCTGCACAA length is 16nt, SEQ ID NO: 4;
  • GTGCAGACTCCGGTGG (length is 16nt, SEQ ID NO: 7);
  • CTTTATAAACAACCAG (16 nt in length, SEQ ID NO: 8).
  • C2 circular single-stranded DNA
  • B12 circular double-stranded DNA template with a bubble structure length of 12 bp
  • reaction system and conditions for preparing double-stranded RNA with B12 and conditions for electrophoresis detection of preparation results Same as embodiment 1, no more details here.
  • C in Figure 5 is the experimental result. It can be seen that after adding RNase H for secondary digestion, the smeared bands above the lane are reduced, and the amount of short products at the lower position of the lane is increased. This suggests that the addition of ssDNA did not play a role (if it did, the product would be cleaved into oligonucleotides), but that the newly added RNase H recognized the DNA produced by the hybridization of the ssDNA at the vesicle to the RNA product /RNA chimera and further played the role of enzyme digestion. The above results show that the transcript of B12 is double-stranded, not single-stranded.
  • ACCGGAGTCTGCACAA length is 16nt, SEQ ID NO: 4;
  • GTGCAGACTCCGGTGG (length is 16nt, SEQ ID NO: 7);
  • CTTTATAAACAACCAG (16 nt in length, SEQ ID NO: 8).
  • ACCGGAGTCTGCACAA length is 16nt, SEQ ID NO: 4;
  • GTGCAGACTCCGGTGG (length is 16nt, SEQ ID NO: 7);
  • CTTTATAAACAACCAG (16 nt in length, SEQ ID NO: 8).
  • C in Figure 7 is the experimental result, and it can be seen that the results are basically consistent with C in Figure 4, so we judge that the transcription product of B5 is double-stranded RNA.
  • the size of the bubble structure is 5 bp, it can be seen that when T7 RNAP and RNase H coexist in the transcription system, there is no fixed-length short RNA product after transcription, but only diffuse bands appear. Most of the products are still concentrated in the gel pores, indicating that such a small size of the bubble structure limits the enzymatic digestion and separation of transcripts by RNase H, making the separation of product monomers unsatisfactory. Therefore, the size of the bubble structure should be ⁇ 5bp when designing the transcription template.
  • ACCGGAGTCTGCACAA length is 16nt, SEQ ID NO: 4;
  • GTGCAGACTCCGGTGG (length is 16nt, SEQ ID NO: 7);
  • CTTTATAAACAACCAG (16 nt in length, SEQ ID NO: 8).
  • C in Figure 8 is the experimental result, and it can be seen that the result is similar to that of C in Figure 5.
  • the diffuse band above the swimming lane is reduced, and the amount of the short product at the lower position of the swimming lane is reduced. greatly improved.
  • the presence of polyT at the vesicular structure greatly affects the digestion and separation of transcripts by RNase H. Such special sequences should be avoided as much as possible.
  • ACCGGAGTCTGCACAA length is 16nt, SEQ ID NO: 4;
  • GTGCAGACTCCGGTGG (length is 16nt, SEQ ID NO: 7);
  • CTTTATAAACAACCAG (16 nt in length, SEQ ID NO: 8).
  • TGATTCCTGGACCTTTTGTG length is 20nt, SEQ ID NO: 17;
  • CACAAAAGGTCCAGGAATCA length is 20nt, SEQ ID NO: 20;
  • CTCACATCTTCGACTCCTGT (20 nt in length, SEQ ID NO: 21).
  • the preparation method of the 96nt single circle is the same as in Example 1, but the preparation method of the 96bp circular double-stranded DNA template is different from that of Examples 1-6. Specifically: the prepared 96nt single loop and the two short DNA strands (A1 and B1) corresponding to its complementary strands were annealed and hybridized (the annealing conditions were the same as in Example 1), and Taq Dn1 was directly added for connection. The purification method of the 96bp circular double-stranded DNA template and the subsequent transcription conditions are the same as in Example 1, and will not be repeated here.
  • Figure 10 is the result of preparing double-stranded RNA with a circular double-stranded DNA template with a length of 96 bp. The results showed that the transcripts were also double-stranded RNA, indicating that the method had good generality.
  • GAGTTTCTTCACCTTTTGTG (20 nt in length, SEQ ID NO: 23);
  • TGATTCCTGGTTGATGAAAG length is 20nt, SEQ ID NO: 24;
  • CTTTCATCAACCAGGAATCA (20nt in length, SEQ ID NO: 26);
  • CACAAAAGGTGAAGAAACTC (length is 20nt, SEQ ID NO: 27);
  • CTCACATCTTCGACTCCTGT (20 nt in length, SEQ ID NO: 21).
  • the method for preparing the 155nt single ring is different from the one-pot method in Example 1, and is prepared in a step-by-step manner. Specifically, L-A1B1 was prepared first, and then C-A1B1C1 (155 nt single ring) was prepared using L-A1B1 and C1 as raw materials.
  • T4 DNA ligase T4 Dnl, final concentration of 0.125 U / ⁇ L was mixed with the corresponding T4 DNA ligase buffer (final concentration: 0.5 ⁇ ), and reacted at 37°C for 2h.
  • the preparation method of the 155bp circular double-stranded DNA template is the same as in Example 7. Specifically: the prepared 155nt single loop and three short DNA strands (A, B and C) corresponding to its complementary strands were annealed and hybridized (the annealing conditions were the same as in Example 1), and Taq Dn1 was directly added for connection.
  • the purification method of the 155bp circular double-stranded DNA template and the subsequent transcription conditions are the same as in Example 1, and will not be repeated here.
  • Figure 11 is the result of preparing double-stranded RNA with a circular double-stranded DNA template with a length of 155 bp. The results showed that the transcripts were also double-stranded RNA, indicating that the method had good generality.
  • GAGTTTCTTCACCTTTTGTG (20 nt in length, SEQ ID NO: 23);
  • GTGGTAGCCGTCGGCGGACA length is 20nt, SEQ ID NO: 33;
  • CACAAAAGGTGAAGAAACTC (length is 20nt, SEQ ID NO: 27);
  • CTTTCATCAAAATCGGCAGT (20nt in length, SEQ ID NO: 38);
  • ACTCCAACGCACTCAGTACG length is 20nt, SEQ ID NO: 39;
  • TGTCCGCCGACGGCTACCAC length is 20nt, SEQ ID NO: 40;
  • the preparation method of the 278nt single ring is the same as in Example 8, that is, it is prepared in a step-by-step manner. Specifically, L-B1C1 and L-D1E1 were prepared first, then L-D1E1F1 was prepared from L-D1E1 and F1, and finally C-B1C1D1E1F1 (278nt single ring) was prepared from L-B1C1 and L-D1E1F1.
  • the prepared L-D1E1 (final concentration is 2 ⁇ M), F1 (final concentration is 2 ⁇ M), splint (Splint F1E1, final concentration is 3 ⁇ M), T4 DNA ligase (T4 Dnl, final concentration is 0.125 U/ ⁇ L) and The corresponding T4 DNA ligase buffer (final concentration: 0.5 ⁇ ) was mixed and reacted at 37°C for 2 hours.
  • L-D1E1F1 final concentration of 1 ⁇ M
  • L-B1C1 final concentration of 1 ⁇ M
  • splint Splint D1C1 and Splint B1F1, final concentration of 1.5 ⁇ M
  • T4 DNA ligase T4 Dnl, final concentration 0.0625U/ ⁇ L
  • T4 DNA ligase buffer final concentration: 0.25 ⁇
  • the preparation method of the circular double-stranded DNA template of 278 bp is the same as that in Example 7 and Example 8. Specifically: the prepared 278nt single circle and five short DNA strands (B, C, D, E and F) corresponding to its complementary strands were annealed and hybridized (the annealing conditions were the same as in Example 1) and directly added to Taq Dn1 for connection. The purification method of the 278bp circular double-stranded DNA template and the subsequent transcription conditions are the same as in Example 1, and will not be repeated here.
  • Fig. 12 is the result of preparing double-stranded RNA with a circular double-stranded DNA template with a length of 278bp. The results showed that the transcripts were also double-stranded RNA, indicating that the method had good generality.
  • Example 1 The raw materials used, the preparation and purification methods of single-circle, single-stranded and circular double-stranded DNA are the same as those in Example 1, and will not be repeated here. The difference is that SP6 RNA polymerase is used to transcribe here, and the transcription conditions are the same as in Example 1.
  • Figure 13 is the result of B15 preparing double-stranded RNA under the action of SP6 RNA polymerase. The results showed that the transcripts were also double-stranded RNA, indicating that this method has good generality and is applicable to many common RNA polymerases.

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Abstract

一种制备双链RNA的方法，主要解决现有双链RNA制备方法步骤繁琐、效率低、成本高昂且难以实现大量制备的问题。针对要制备的双链RNA，先设计一个含有长度为5-15bp泡状结构的、无启动子的环状双链DNA模板；然后在体系中同时加入上述无启动子环状双链DNA模板、RNA聚合酶、核糖核酸酶H、RNA酶抑制剂、NTPs和无机焦磷酸酶进行转录，转录过程中RNA聚合酶进行无偏好性的高效双向滚环转录，核糖核酸酶H则在环状双链DNA模板中泡状结构处单链DNA的辅助下实现对含有重复序列的转录产物的酶切，获得满足要求的双链RNA单体。所述方法实现了双链RNA的一步、高效、大量制备。

Description

一种制备双链RNA的方法 技术领域
本发明属于核酸技术领域，具体涉及一种制备双链RNA的方法。
背景技术
双链RNA(dsRNA)结构在自然界中普遍存在，其常作为长链RNA，如核糖体RNA(rRNA)、信使RNA(mRNA)以及类病毒(一种仅由RNA组成的病毒)等的一小部分。长度为几百～几千个碱基对的长双链RNA，如pre-miRNA(其由mRNA的一部分和与其互补的RNA链组成)和双链RNA病毒等也被发现广泛存在。其中最有前景的应用之一是以小干扰RNA(small interfering RNAs，siRNAs)为工具来发挥作用的RNA干扰(RNA interference,RNAi)技术，其是用于转录后基因调控的有力工具(Nature Genet,2002,32,107-108；EMBO J,2001,20,6877-6888；Proc Natl Acad Sci USA,2002,99,9942-9947.)。近年来RNAi技术被尝试应用于农业(病虫害防治)(Insect Sci,2013,20,4-14；Pest Manag Sci,2016,72,801-809)和水产养殖业(虾病防治)(J Biotechnol,2020,321,48-56；J Virol Methods,2013,188,64-69；IOP Conf Ser Earth Environ Sci,2020,584,012051.)中。但目前dsRNA的大量、高效合成尚未实现，这在很大程度上制约了RNAi农(渔)药、RNAi疫苗的推广使用。因此，亟需建立一种可满足dsRNA高效、低成本、简易制备的方法，以满足科研需求和市场需求。
dsRNA在体内/外均可制备(Anal Bioanal Chem,2018,410,3239-3252)。体内制备(重组过表达)时需要先将用于转录的模板重组到质粒载体当中，然后将其导入大肠杆菌(E.coli)(J.Biotechnol,2020,321,48-56；J Virol Methods,2013,188,64-69；IOP Conf.Ser.Earth Environ Sci,2020,584,012051.)、植物细胞(UK Patent,No.WO2020183416-A1,Sep17,2020)或哺乳动物细胞(Methods Mol Biol,2013,942,291-314.)中进行诱导培养。整个过程需经历表达载体构建、转染、细胞培养、裂解、产物提取纯化(需去除宿主自身的rRNA等杂质)等诸多步骤，较为繁琐；并且胞内机质复杂，产物易被核酸酶降解的同时生物安全性也难以保证。目前这些体内制备技术尚处于研究阶段，未被用于实际生产。
体外制备方法可分为化学合成和酶法合成(转录)两大类。化学合成法(固相合成)存在合成步骤繁琐(EMBO J,2001,20,6877-6888；Nat Struct Biol,1998,5,203-212；Isr J Chem,2013,53,326-349)、产物纯度较差(Anal Biochem,2001,298,196-206；Chin J Biotech,2018,34,664)、难以实现大量合成、生物安全性较差(使用多种有机试剂)(Chin J  Biotech,2018,34,664)等诸多缺陷。相较化学合成而言，酶法因在液相环境中进行而更易于实现大规模生产，且由于在整个过程中没有涉及到有害有机试剂，因此产品的安全性更好(Isr J Chem,2013,53,326-349；Chimia,2005,59,812-816)。近些年的研究还表明酶法可满足带修饰RNA的合成，发展前景良好(Org Biomol Chem,2018,16,5800-5807；Molecules,2020,25,5492)。按照模板为线性还是环状，现有dsRNA酶法合成方法可分为线性转录和滚环转录。线性转录可分为两种情况，一种是用两种线性双链DNA模板分别转录出sense链和antisense链，然后退火杂交获得dsRNA(Nucleic Acids Res,2002,30,e46；Nucleic Acids Res,2003,31,e38)；另一种是直接用含有回文序列的线性双链DNA模板制备得到同时含有sense序列和antisense序列的发卡型结构的RNA，再由体内的Dicer酶加工处理得到目标siRNA(Mol Biotechnol,2017,59,73-83)。上述两种方法中所用到的线性双链DNA模板中均包含启动子序列，且为了能高效起始转录，有些模板中启动子下游的最初两个碱基须为GG，要想得到精确的产物，后续需酶切去除多余转录出的CC(Nucleic Acids Res,2003,31,e38)。由此可见上述线性转录法均较繁琐，且效率低下，难以满足实际应用需求。相较线性转录而言，滚环转录的效率要高出许多。现有滚环转录法也可分为两类。一类是直接用一个环状单链DNA(哑铃环(Oligonucleotides,2006,16,353-363)或“Y”型单环(Nano Lett,2018,18,4279-4284))同时转录出sense序列和antisense序列。这种方法虽较简便，但需对模板进行加长设计，这样就会额外转录出无用序列，需后续纯化。还有一类是用两种环状单链DNA分别转录出sense序列和antisense序列，再退火杂交得到目的dsRNA(Sci Rep,2017,7,10005；Adv Sci(Weinh),2017,4,1600523)。采用这种方法时，由于环状单链DNA的转录效率易受模板序列和自身二级结构的影响(J Am Chem Soc,1995,117,7818-7819；Proc Natl Acad Sci USA,2002,99,54-59；Nucl Acids Res,2013,41,2552-2564；Nucl Acids Res,2014,42,10596-10604；Science Advance Today,2015,25226.)，可能会发生不对称转录(一个环状单链DNA转录效率高，一个转录效率低)，使得产物为dsRNA和ssRNA(单链RNA)的混合物。由此可见现有方法均难以实现dsRNA的大量、高效合成。
发明内容
针对现有双链RNA制备方法步骤繁琐、效率低、成本高昂且难以实现大量制备的问题，本发明提供了一种制备双链RNA的方法，针对要制备的双链RNA，先设计一个含有5-15bp泡状结构(Bubble)的、无启动子的环状双链DNA模板；然后在体系中同时加入上述环状双链DNA模板、RNA聚合酶、核糖核酸酶H、NTPs、RNA酶抑制剂、无机焦磷酸酶进行转录。在转录过程中RNA聚合酶进行无偏好性的高效双向滚环转录，RNase H则在该 模板中泡状结构处未杂交的DNA部分的辅助下及时实现对转录产物的酶切。在RNA聚合酶、核糖核酸酶H和含泡状结构无启动子环状双链DNA模板三者之间的默契配合下实现了目的双链RNA产物的一步、高效、大量制备。该方法非常简便高效，低成本，可操作性较强，适用于工业化大规模生产。
本发明采用的技术方案如下：
一种制备双链RNA的方法，包括以下步骤：
步骤一，设计并制备环状双链DNA模板，所述环状双链DNA模板含有不互补的泡状结构部分和互补部分，不含启动子序列，所述互补部分与要制备的双链RNA长度相同且序列相同，序列中T对应U，所述泡状结构处于环状双链DNA模板中互补部分的两端，泡状结构的长度为5-15bp，泡状结构的两条链之间连续互补的碱基不超过2个；
步骤二，将制备的环状双链DNA模板纯化，去除环状双链DNA模板中混杂着的线性单链DNA；
步骤三，将纯化的环状双链DNA模板、RNA聚合酶、核糖核酸酶H、NTPs、RNA酶抑制剂、无机焦磷酸酶和相应的RNA聚合酶缓冲液混合，进行转录，所述RNA聚合酶为T7 RNA聚合酶、SP6 RNA聚合酶或者T3 RNA聚合酶，在转录过程中RNA聚合酶进行无偏好性的高效双向滚环转录，核糖核酸酶H则在环状双链DNA模板中泡状结构处单链DNA的辅助下及时实现对含有重复序列的转录产物的酶切，获得满足要求的目的双链RNA单体。
泡状结构的上、下链的序列相同或不同均可。泡状结构处的上、下链中均应避免形成发卡结构或polyA、polyT等序列。
泡状结构尺寸应在5-15碱基之间，否则会导致双链RNA的产量降低。
不考虑环状双链DNA模板制备的难易程度，所述本发明可制备的双链RNA产物单体的长度为70bp以上；优选70-1000bp，再优选为90-437bp，更优选为96-278bp。
进一步的，当所述环状双链DNA模板长度小于90bp时，采用一段法，设计一条成环前体线性链制备环状单链DNA，环状单链DNA和线性单链DNA退火杂交后加入DNA连接酶和对应的连接酶缓冲液进行连接反应；
当所述环状双链DNA模板长度大于90bp时，采用一锅法或分步法制备环状单链DNA，然后将环状单链DNA和其互补链所对应的多条短DNA链混合，退火杂交后加入DNA连接酶和对应的连接酶缓冲液进行连接反应；
所述环状单链DNA和线性单链DNA杂交后，线性单链DNA的两端距离泡状结构的边缘应大于15nt。设计成环前体线性链时，需避免将断点设计在泡状结构处，否则在制备环状双 链DNA模板的退火杂交过程中无法形成Nick(连接酶发挥连接作用所必需的结构)。
进一步的，所述制备环状双链DNA模板的方法，当所述环状双链DNA模板长度为70-90bp时，采用一步法环化制备所述环状单链DNA；当所述环状双链DNA模板长度大于90bp时，设计多段40-90nt的
Figure PCTCN2022092023-appb-000001
磷酸化的DNA片段和相应的splint，采用一锅法，进行一步或分步连接反应制备环状单链DNA或线性单链DNA。
进一步的，所述退火杂交条件为：先在90℃下保温1-3min，然后以0.1℃/s的速度降温至20-25℃并保温10min。
进一步的，当使用T4DNA连接酶和相应的T4DNA连接酶缓冲液时，在4-37℃范围内保温2-24h进行连接；当使用TaqDNA连接酶和相应的TaqDNA连接酶缓冲液时，在45-80℃范围内保温2-24h进行连接。
进一步的，当使用T4DNA连接酶时，采用终浓度为0.05-1×的缓冲液，当缓冲液浓度为1×时，其组成为：40mM Tris-HCl，10mM MgCl 2，10mM DTT，500μM ATP(pH 7.8@25℃)；当使用Taq DNA连接酶时，采用终浓度为1×的缓冲液，其组成为：20mM Tris-HCl，25mM KAc，10mM Mg(Ac) 2，10mM DTT，1.0mM NAD，0.1％Triton X-100(pH 7.6@25℃)。
进一步的，所述步骤二的环状双链DNA模板的纯化过程中，用Exo I和Exo III共同酶切去除环状双链DNA模板中混杂着的所有线性单链DNA，Exo I的酶切反应采用终浓度为0.5×的缓冲液，其组成为：33.5mM glycine-KOH(pH 9.5@25℃)，3.35mM MgCl 2，0.5mM DTT；Exo III的酶切反应采用终浓度为0.5×的缓冲液，其组成为：33mM Tris-HCl(pH 8.0@30℃)，0.33mM MgCl 2。
进一步的，所述步骤三在37℃下恒温反应0.5-72h制备双链RNA。
进一步的，双链RNA的制备过程中，RNA聚合酶的转录反应采用终浓度为1×的缓冲液，其组成为：40mM Tris-HCl(pH 7.9@25℃)，6.0mM MgCl 2，10mM DTT，10mM NaCl和2mM spermidine。
有益效果：
本发明制备双链RNA的方法中所用到的转录模板中无需引入启动子序列，因而转录产物中不会引入由启动子转录产生的多余序列，极大方便了后续的产物纯化过程。
双向转录效率相同，避免了非对称转录导致的一条RNA链过量的情况的发生，特别适合制备双链RNA。
相较于现有的双链RNA制备方法，本发明所涉及的制备双链RNA的方法非常简便、 易操作，无需严苛条件，仅需扩大反应体系、延长转录时间、供给转录体系充足的NTPs即可实现目的双链RNA产物的大量制备。
利用本方法制备得到的双链RNA本身纯度较高(仅首尾两端的2-8nt为单链RNA)，若对产物纯度要求较高，后续可用RNase A/RNase 1/RNase T1等酶切去除首尾两端的单链RNA，用DNase I等DNA酶去除环状双链DNA模板，用抽提醇沉、HPLC等方式对产物进一步纯化。
综上，本发明大大简化了双链RNA的制备流程，具有良好的应用潜力。
附图说明
图1实施例1制备双链RNA的示意图；
图2实施例1环状单链DNA的制备方法示意图；
图3实施例1环状双链DNA模板的制备方法示意图；
图4实施例1用B15制备双链RNA的结果(用T7 RNA聚合酶制备)，其中A为B15的序列设计示意图，B为B15在75℃下制备的结果示意图；C为用B15制备双链RNA的结果以及对转录产物的酶切验证结果；
图5实施例2用B12制备双链RNA的结果，其中A为B12的序列设计示意图，B为B12在75℃下制备的结果示意图；C为用B12制备双链RNA的结果以及对转录产物的酶切验证结果；
图6实施例3用B9制备双链RNA的结果，其中A为B9的序列设计示意图，B为B9在75℃下制备的结果示意图；C为用B9制备双链RNA的结果以及对转录产物的酶切验证结果；
图7实施例4用B5制备双链RNA的结果，其中A为B5的序列设计示意图，B为B5在75℃下制备的结果示意图；C为用B5制备双链RNA的结果以及对转录产物的酶切验证结果；
图8实施例5用B9T制备双链RNA的结果，其中A为B9T的序列设计示意图，B为B9T在75℃下制备的结果示意图；C为用B9T制备双链RNA的结果以及对转录产物的酶切验证结果；
图9实施例6用B5T制备双链RNA的结果，其中A为B5T的序列设计示意图，B为B5T在75℃下制备的结果示意图；C为用B5T制备双链RNA的结果以及对转录产物的酶切验证结果；
图10实施例7用环96制备双链RNA的结果，其中A为环96的序列设计示意图，B为环96在65℃下制备的结果示意图；C为用环96制备双链RNA的结果以及对转录产物的酶切验证结果；
图11实施例8用环155制备双链RNA的结果，其中A为环155的序列设计示意图，B为环155在65℃下制备的结果示意图；C为用环155制备双链RNA的结果以及对转录产物的酶切验证结果；
图12实施例9用环278制备双链RNA的结果，其中A为环278的序列设计示意图，B为环278在65℃下制备的结果示意图；C为用环278制备双链RNA的结果以及对转录产物的酶切验证结果；
图13实施例10用B15制备双链RNA的结果(用SP6 RNA聚合酶制备)。
具体实施方式
下面结合具体实施例及附图对本发明做进一步详细说明。
下述实施例使用的用于制备环状双链DNA模板的成环前体线性链均购自生工生物工程(上海)股份有限公司，为人工合成；核糖核酸酶H(RNase H)、10×RNase H buffer、Taq DNA连接酶(Taq Dnl)、10×Taq Dnl buffer、无机焦磷酸酶(Inorganic Pyrophosphatase,E.coli)、ShortCut RNase III及其对应的10×buffer购自安诺伦(北京)生物科技有限公司(NEW ENGLAND BioLabs)；T7 RNA聚合酶(T7 RNAP)、SP6 RNA聚合酶(SP6 RNAP)、5×RNAP buffer、核酸酶抑制剂(RiboLock RNase Inhibitor)、T4多聚磷酸激酶(T4 PNK)、10×T4 PNK buffer A、ATP(原液浓度为100mM)、T4 DNA连接酶(T4 Dnl)、10×T4 Dnl buffer、Exo I、Exo I对应的10×buffer、Exo III、Exo III对应的10×buffer、DNase I、10×DNase I buffer(含Mg 2+)、NTPs(ATP、UTP、CTP和GTP的统称，原液浓度为100mM)均购自美国赛默飞世尔生物科技有限公司；核酸染液(Ultra GelRed)购自诺唯赞(南京)生物科技有限公司；其他化学用品购自美国西格玛奥德里奇(Sigma-Aldrich)。
以下实施例是为了证明本发明的方法具有优越性。
实施例1
(1)原料
A-a链
Figure PCTCN2022092023-appb-000002
ACTCCGGTGGAATGAAGGACCAAGTCTGTCATGCACTGAAATCAGTCTCATTGCTTTATAA(
Figure PCTCN2022092023-appb-000003
-磷酸化，长度为61nt，SEQ ID NO：1)；
A-b链
Figure PCTCN2022092023-appb-000004
ACAACCAGCTAAGACACTGCCATACCCTGTAGAACCGAATTTGTGCAG(
Figure PCTCN2022092023-appb-000005
-磷酸化，长度为48nt，SEQ ID NO：2)；
Splint A-ab
Figure PCTCN2022092023-appb-000006
AGCTGGTTGTTTATAAAGCA(长度为20nt，SEQ ID NO：3)；
Splint A-ba
Figure PCTCN2022092023-appb-000007
ACCGGAGTCTGCACAA(长度为16nt，SEQ ID NO：4)；
C1-a链
Figure PCTCN2022092023-appb-000008
TTATAAAGCAATGAGACTGATTTCAGTGCATGACAGACTTGGTCCTTCATTCCACCGGA(
Figure PCTCN2022092023-appb-000009
-磷酸化，长度为59nt，SEQ ID NO：5)；
C1-b链
Figure PCTCN2022092023-appb-000010
GTCTGCACAAATTCGGTTCTACACCCATACCGTCACAGTTAGCTGGTTGT(
Figure PCTCN2022092023-appb-000011
-磷酸化，长度为50nt，SEQ ID NO：6)；
Splint C1-ab
Figure PCTCN2022092023-appb-000012
GTGCAGACTCCGGTGG(长度为16nt，SEQ ID NO：7)；
Splint C1-ba
Figure PCTCN2022092023-appb-000013
CTTTATAAACAACCAG(长度为16nt，SEQ ID NO：8)。
(2)制备环状单链DNA(C1)及线性单链DNA(LA)
C1的制备：
将成环前体线性链(C1-a和C1-b，终浓度为1μM)、splint(Splint C1-ab和Splint C1-ba，终浓度为2μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.125U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为1×)混合，37℃下反应12h制备C1。
1×T4 Dnl Buffer的组成：40mM Tris-HCl，10mM MgCl 2，10mM DTT，500μM ATP(pH 7.8@25℃)。
LA的制备：
将前体线性链(A-a和A-b，终浓度为1μM)、splint(Splint A-ab，终浓度为2μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.125U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为1×)混合，37℃下反应12h制备LA。
1×T4 Dnl Buffer的组成：40mM Tris-HCl，10mM MgCl 2，10mM DTT，500μM ATP(pH 7.8@25℃)
(3)制备B15(泡状结构长度为15bp的环状双链DNA模板)
先将C1(终浓度为1μM)、LA(终浓度为1.2μM)以及Taq Dnl buffer(终浓度为1×)混的Mix在90℃下保温1～3min，然后以0.1℃/s的速度降温至60℃，再以0.1℃/s的速度降 温至20-25℃并保温10min使得C1和LA充分杂交形成nick。将PCR调到75℃下运行，然后在样品中加入Taq Dnl(终浓度为4U/μL)在75℃下连接12h。
1×Taq Dnl Buffer的组成：20mM Tris-HCl，25mM KAc，10mM Mg(Ac) 2，10mM DTT，1mM NAD，0.1％Triton X-100(pH 7.6@25℃)。
(4)纯化B15
在(3)中制备得到的B15中加入Exo I(终浓度约为1U/μL)、Exo III(终浓度约为4U/μL)及其对应的buffer(各自终浓度均为0.5×)。颠倒混匀并离心后在37℃下过夜酶切，以去除体系中剩余的LA以及splint。
0.5×Exo I buffer的组成为：33.5mM glycine-KOH(pH 9.5@25℃)，3.35mM MgCl 2，0.5mM DTT；
0.5×Exo III buffer的组成为：33mM Tris-HCl(pH 8.0@30℃)，0.33mM MgCl 2。
(5)用B15转录制备双链RNA
转录体系及反应条件(100μL)：[B15]＝50nM，[each NTP]＝0.5mM，[T7 RNAP]＝2U/μL，[RNase Inhibitor]＝2U/μL，[RNAP buffer]＝1×，([RNase H])＝0.25U/μL。在37℃下转录12h，再在70℃下处理10min终止反应。
1×RNAP buffer的组成为：40mM Tris-HCl(pH 7.9@25℃)，6mM MgCl 2，10mM DTT，10mM NaCl和2mM spermidine。
(6)用ShortCut RNase III酶切转录产物以确认转录产物为双链：
取出10μL转录产物，然后在体系中(总体积为20μL)加入ShortCut RNase III(终浓度为0.2U/μL)、ShortCut Reaction buffer(终浓度为1×)和MnCl 2(终浓度为1×)。在37℃下酶切40min，然后取出部分酶切产物与loading buffer混合(loading buffer中有EDTA，可以螯合掉金属离子，从而终止反应)。
(7)电泳检测
对实验结果进行8％尿素变性聚丙烯酰胺凝胶电泳检测，再用Image Lab软件分析环状双链DNA模板的制备结果、其对应的双链RNA的制备结果(转录结果)以及ShortCut RNase III的转录产物酶切结果。
图4为结果图，其中图4中A为B15的序列设计示意图，其含有一个上、下链序列相同、方向相反的15bp泡状结构；图4中B为B15在75℃下制备的结果示意图；图4中C为用B15制备双链RNA(体系中仅有T7 RNAP或T7 RNAP和RNase H共存)的结果以及ShortCut RNase III对转录产物的酶切验证结果，图中的标注“S”指ShortCut RNase III酶切 的结果。结果表明当转录体系中仅有T7 RNAP时，转录产物为堵在胶孔处的多聚串联重复序列；当T7 RNAP和RNase H共存于转录体系时，转录产物集中分布于一个固定的长度；ShortCut RNase III酶切后，上述转录产物均被切短了，长度集中在一小段固定区域，根据该酶的酶切特性(其可将长双链RNA酶切成18-25bp的短双链RNA)，可以判定B15的转录产物为我们想要的双链RNA。
实施例2
(1)原料
A-a链
Figure PCTCN2022092023-appb-000014
ACTCCGGTGGAATGAAGGACCAAGTCTGTCATGCACTGAAATCAGTCTCATTGCTTTATAA(
Figure PCTCN2022092023-appb-000015
-磷酸化，长度为61nt，SEQ ID NO：1)；
A-b链
Figure PCTCN2022092023-appb-000016
ACAACCAGCTAAGACACTGCCATACCCTGTAGAACCGAATTTGTGCAG(
Figure PCTCN2022092023-appb-000017
-磷酸化，长度为48nt，SEQ ID NO：2)；
Splint A-ab
Figure PCTCN2022092023-appb-000018
AGCTGGTTGTTTATAAAGCA(长度为20nt，SEQ ID NO：3)；
Splint A-ba
Figure PCTCN2022092023-appb-000019
ACCGGAGTCTGCACAA(长度为16nt，SEQ ID NO：4)；
C2-a链(
Figure PCTCN2022092023-appb-000020
同C1-a)：
TTATAAAGCAATGAGACTGATTTCAGTGCATGACAGACTTGGTCCTTCATTCCACCGGA(
Figure PCTCN2022092023-appb-000021
-磷酸化，长度为59nt，SEQ ID NO：5)；
C2-b链
Figure PCTCN2022092023-appb-000022
GTCTGCACAAATTCGGTTCTACACCCATACCGTCAGTCTTAGCTGGTTGT(
Figure PCTCN2022092023-appb-000023
-磷酸化，长度为50nt，SEQ ID NO：9)；
Splint C1-ab
Figure PCTCN2022092023-appb-000024
GTGCAGACTCCGGTGG(长度为16nt，SEQ ID NO：7)；
Splint C1-ba
Figure PCTCN2022092023-appb-000025
CTTTATAAACAACCAG(长度为16nt，SEQ ID NO：8)。
环状单链DNA(C2)的制备、B12(泡状结构长度为12bp的环状双链DNA模板)的制备及纯化、用B12制备双链RNA的反应体系及条件以及电泳检测制备结果的条件同实施例1，此处不再赘述。
(2)转录结束后在转录产物中加入A链或C2链的一部分与之杂交，然后加入 RNase H进行二次酶切以判断转录产物为单/双链的体系及反应条件：
在10μL转录产物中加入LC2-a(LC1-a)/LA-a(终浓度为0.1μM)和RNase H buffer(终浓度为0.5×)，然后在65℃下处理10min，再以0.1℃/s的速度降温至37℃，再在37℃下保持5min，接着加入RNase H(终浓度为0.25U/μL)，在37℃下酶切40min。反应结束后在70℃下处理10min，以灭活RNase H。
图5中C为实验结果，可以看到加入RNase H进行二次酶切后泳道上方的弥散条带减少了，且泳道靠下位置处的短产物量得到了提高。这表明加入的单链DNA并未发挥作用(其若发挥作用，产物会被切成寡核苷酸，而是新加入的RNase H识别了泡状结构处的单链与RNA产物杂交产生的DNA/RNA chimera并进一步发挥了酶切作用。上述结果说明B12的转录产物为双链，不是单链。
实施例3
(1)原料
A-a链
Figure PCTCN2022092023-appb-000026
ACTCCGGTGGAATGAAGGACCAAGTCTGTCATGCACTGAAATCAGTCTCATTGCTTTATAA(
Figure PCTCN2022092023-appb-000027
-磷酸化，长度为61nt，SEQ ID NO：1)；
A-b链
Figure PCTCN2022092023-appb-000028
ACAACCAGCTAAGACACTGCCATACCCTGTAGAACCGAATTTGTGCAG(
Figure PCTCN2022092023-appb-000029
-磷酸化，长度为48nt，SEQ ID NO：2)；
Splint A-ab
Figure PCTCN2022092023-appb-000030
AGCTGGTTGTTTATAAAGCA(长度为20nt，SEQ ID NO：3)；
Splint A-ba
Figure PCTCN2022092023-appb-000031
ACCGGAGTCTGCACAA(长度为16nt，SEQ ID NO：4)；
C3-a链(
Figure PCTCN2022092023-appb-000032
同C1-a)：
TTATAAAGCAATGAGACTGATTTCAGTGCATGACAGACTTGGTCCTTCATTCCACCGGA(
Figure PCTCN2022092023-appb-000033
-磷酸化，长度为59nt，SEQ ID NO：5)；
C3-b链
Figure PCTCN2022092023-appb-000034
GTCTGCACAAATTCGGTTCTACACCCATACCGAGTGTCTTAGCTGGTTGT(
Figure PCTCN2022092023-appb-000035
-磷酸化，长度为50nt，SEQ ID NO：10)；
Splint C1-ab
Figure PCTCN2022092023-appb-000036
GTGCAGACTCCGGTGG(长度为16nt，SEQ ID NO：7)；
Splint C1-ba
Figure PCTCN2022092023-appb-000037
CTTTATAAACAACCAG(长度为16nt，SEQ ID NO：8)。
环状单链DNA(C3)的制备、B9(泡状结构长度为9bp的环状双链DNA模板)的制备及纯化、用B9制备双链RNA的反应体系及条件、B9转录产物的ShortCut RNase III酶切表征实验以及电泳检测制备结果的条件同实施例1，此处不再赘述。
图6中C为实验结果，可以看到结果同图4中C基本一致，因此我们判定B9的转录产物为双链RNA。
实施例4
(1)原料
A-a链
Figure PCTCN2022092023-appb-000038
ACTCCGGTGGAATGAAGGACCAAGTCTGTCATGCACTGAAATCAGTCTCATTGCTTTATAA(
Figure PCTCN2022092023-appb-000039
-磷酸化，长度为61nt，SEQ ID NO：1)；
A-b链
Figure PCTCN2022092023-appb-000040
ACAACCAGCTAAGACACTGCCATACCCTGTAGAACCGAATTTGTGCAG(
Figure PCTCN2022092023-appb-000041
-磷酸化，长度为48nt，SEQ ID NO：2)；
Splint A-ab
Figure PCTCN2022092023-appb-000042
AGCTGGTTGTTTATAAAGCA(长度为20nt，SEQ ID NO：3)；
Splint A-ba
Figure PCTCN2022092023-appb-000043
ACCGGAGTCTGCACAA(长度为16nt，SEQ ID NO：4)；
E-a链(
Figure PCTCN2022092023-appb-000044
同C1-a)：
TTATAAAGCAATGAGACTGATTTCAGTGCATGACAGACTTGGTCCTTCATTCCACCGGA(
Figure PCTCN2022092023-appb-000045
-磷酸化，长度为59nt，SEQ ID NO：5)；
E-b链
Figure PCTCN2022092023-appb-000046
GTCTGCACAAATTCGGTTCTACACCCATTGGCAGTGTCTTAGCTGGTTGT(
Figure PCTCN2022092023-appb-000047
-磷酸化，长度为50nt，SEQ ID NO：11)；
Splint C1-ab
Figure PCTCN2022092023-appb-000048
GTGCAGACTCCGGTGG(长度为16nt，SEQ ID NO：7)；
Splint C1-ba
Figure PCTCN2022092023-appb-000049
CTTTATAAACAACCAG(长度为16nt，SEQ ID NO：8)。
环状单链DNA(CE)的制备、B5(泡状结构长度为5bp的环状双链DNA模板)的制备及纯化、用B5制备双链RNA的反应体系及条件、B5转录产物的ShortCut RNase III酶切表征实验以及电泳检测制备结果的条件同实施例1，此处不再赘述。
图7中C为实验结果，可以看到结果同图4中C基本一致，因此我们判定B5的转录产物为双链RNA。但应当注意的是，当泡状结构尺寸为5bp时，可以看到当T7 RNAP和RNase H共存于转录体系时，转录结束后并没有固定长度的短RNA产物出现，只是有弥散条带出现，大部分产物仍集中在胶孔处，说明这么小的泡状结构尺寸限制了RNase H对转录产物的酶切分离，使得产物单体的分离效果不理想。因此在设计转录模板时泡状结构尺寸应≥5bp。
实施例5
(1)原料
A-a链
Figure PCTCN2022092023-appb-000050
ACTCCGGTGGAATGAAGGACCAAGTCTGTCATGCACTGAAATCAGTCTCATTGCTTTATAA(
Figure PCTCN2022092023-appb-000051
-磷酸化，长度为61nt，SEQ ID NO：1)；
A-b链
Figure PCTCN2022092023-appb-000052
ACAACCAGCTAAGACACTGCCATACCCTGTAGAACCGAATTTGTGCAG(
Figure PCTCN2022092023-appb-000053
-磷酸化，长度为48nt，SEQ ID NO：2)；
Splint A-ab
Figure PCTCN2022092023-appb-000054
AGCTGGTTGTTTATAAAGCA(长度为20nt，SEQ ID NO：3)；
Splint A-ba
Figure PCTCN2022092023-appb-000055
ACCGGAGTCTGCACAA(长度为16nt，SEQ ID NO：4)；
H2-a链(
Figure PCTCN2022092023-appb-000056
同C1-a)：
TTATAAAGCAATGAGACTGATTTCAGTGCATGACAGACTTGGTCCTTCATTCCACCGGA(
Figure PCTCN2022092023-appb-000057
-磷酸化，长度为59nt，SEQ ID NO：5)；
H2-b链
Figure PCTCN2022092023-appb-000058
GTCTGCACAAATTCGGTTCTACATTTTTTTTTAGTGTCTTAGCTGGTTGT(
Figure PCTCN2022092023-appb-000059
-磷酸化，长度为50nt，SEQ ID NO：12)；
Splint C1-ab
Figure PCTCN2022092023-appb-000060
GTGCAGACTCCGGTGG(长度为16nt，SEQ ID NO：7)；
Splint C1-ba
Figure PCTCN2022092023-appb-000061
CTTTATAAACAACCAG(长度为16nt，SEQ ID NO：8)。
环状单链DNA(CH2)的制备、B9T(带有polyT的泡状结构长度为9bp的环状双链DNA模板)的制备及纯化、用B9T制备双链RNA的反应体系及条件、以及电泳检测制备结果的条件同实施例1，此处不再赘述。
(2)转录结束后在转录产物中加入A链或H2链的一部分与之杂交，然后加入RNase H进行二次酶切以判断转录产物为单/双链的体系及反应条件：
在10μL转录产物中加入LH2-a(LC1-a)/LA-a(终浓度为1μM)和RNase H buffer(终浓度为0.5×)，然后在65℃下处理10min，再以0.1℃/s的速度降温至37℃，再在37℃下处理5min，接着加入RNase H(终浓度为0.25U/μL)，在37℃下酶切2h。反应结束后在70℃下处理10min，以灭活RNase H。
图8中C为实验结果，可以看到结果与图5中C类似，在加入RNase H进行二次酶切后泳道上方的弥散条带减少了，且泳道靠下位置处的短产物量得到了极大提高。这表明加入的单链DNA并未发挥作用，说明B9T的转录产物也为双链。但应当注意的是，与B9的转录结果(图6中C)相比，可以看到泡状结构处polyT的存在很大程度上影响了RNase H对转录产物的酶切分离，在设计转录模板时应尽量避免此类特殊序列。
实施例6
(1)原料
A-a链
Figure PCTCN2022092023-appb-000062
ACTCCGGTGGAATGAAGGACCAAGTCTGTCATGCACTGAAATCAGTCTCATTGCTTTATAA(
Figure PCTCN2022092023-appb-000063
-磷酸化，长度为61nt，SEQ ID NO：1)；
A-b链
Figure PCTCN2022092023-appb-000064
ACAACCAGCTAAGACACTGCCATACCCTGTAGAACCGAATTTGTGCAG(
Figure PCTCN2022092023-appb-000065
-磷酸化，长度为48nt，SEQ ID NO：2)；
Splint A-ab
Figure PCTCN2022092023-appb-000066
AGCTGGTTGTTTATAAAGCA(长度为20nt，SEQ ID NO：3)；
Splint A-ba
Figure PCTCN2022092023-appb-000067
ACCGGAGTCTGCACAA(长度为16nt，SEQ ID NO：4)；
J2-a链(
Figure PCTCN2022092023-appb-000068
同C1-a)：
TTATAAAGCAATGAGACTGATTTCAGTGCATGACAGACTTGGTCCTTCATTCCACCGGA(
Figure PCTCN2022092023-appb-000069
-磷酸化，长度为59nt，SEQ ID NO：5)；
J2-b链
Figure PCTCN2022092023-appb-000070
GTCTGCACAAATTCGGTTCTACATTTTTTGGCAGTGTCTTAGCTGGTTGT(
Figure PCTCN2022092023-appb-000071
-磷酸化，长度为50nt，SEQ ID NO：13)；
Splint C1-ab
Figure PCTCN2022092023-appb-000072
GTGCAGACTCCGGTGG(长度为16nt，SEQ ID NO：7)；
Splint C1-ba
Figure PCTCN2022092023-appb-000073
CTTTATAAACAACCAG(长度为16nt，SEQ ID NO：8)。
环状单链DNA(CJ2)的制备、B5T(带有polyT的泡状结构长度为5bp的环状双链DNA模板)的制备及纯化、用B5T制备双链RNA的反应体系及条件、以及电泳检测制备结果的条件同实施例1，此处不再赘述。
(2)转录结束后在转录产物中加入A链或J2链的一部分与之杂交，然后加入RNase H进行二次酶切以判断转录产物为单/双链的体系及反应条件：
在10μL转录产物中加入LJ2-a(LC1-a)/LA-a(终浓度为1μM)和RNase H buffer(终浓度为0.5×)，然后在65℃下处理10min，再以0.1℃/s的速度降温至37℃，再在37℃下处理5min，接着加入RNase H(终浓度为0.25U/μL)，在37℃下酶切40min。反应结束后在70℃下处理10min，以灭活RNase H。
图9中C为实验结果，可以看到在加入RNase H进行二次酶切后堵在胶孔处的长产物未减少，这表明加入的单链DNA未发挥作用，说明B5T的转录产物也为双链。
实施例7
(1)原料
A链
Figure PCTCN2022092023-appb-000074
CCAGGAATCAGCGGCAAATT CCTCTACTTTCCTCGTCACATCTT(
Figure PCTCN2022092023-appb-000075
-磷酸化，长度为44nt，SEQ ID NO：14，下划线处为泡状结构所在位置)；
B链
Figure PCTCN2022092023-appb-000076
CGACTCCTGTACTGACAACACTCTCACACGACACATCCGCGTCACAAAAGGT(
Figure PCTCN2022092023-appb-000077
-磷酸化，长度为52nt，SEQ ID NO：15)；
Splint AB
Figure PCTCN2022092023-appb-000078
ACAGGAGTCGAAGATGTGAC(长度为20nt，SEQ ID NO：16)；
Splint BA
Figure PCTCN2022092023-appb-000079
TGATTCCTGGACCTTTTGTG(长度为20nt，SEQ ID NO：17)；
A1链
Figure PCTCN2022092023-appb-000080
AAGATGTGA GCTCCTTTCATCTCCAATTTGCCGCTGATTCCTGG(
Figure PCTCN2022092023-appb-000081
-磷酸化，长度为44nt，SEQ ID NO：18，下划线处为泡状结构所在位置)；
B1链
Figure PCTCN2022092023-appb-000082
ACCTTTTGTGACGCGGATGTGTCGTGTGAGAGTGTTGTCAGTACAGGAGTCG(
Figure PCTCN2022092023-appb-000083
-磷酸 化，长度为52nt，SEQ ID NO：19)；
Splint A1B1
Figure PCTCN2022092023-appb-000084
CACAAAAGGTCCAGGAATCA(长度为20nt，SEQ ID NO：20)；
Splint B1A1
Figure PCTCN2022092023-appb-000085
CTCACATCTTCGACTCCTGT(长度为20nt，SEQ ID NO：21)。
96nt的单环制备方法同实施例1，但96bp的环状双链DNA模板的制备方法与实施例1-6均不同。具体地：将制备好的96nt单环和与其互补链对应的两条短DNA链(A1和B1)退火杂交(退火条件同实施例1)后直接加入Taq Dnl进行连接。96bp的环状双链DNA模板的纯化方法及后续的转录条件同实施例1，此处不再赘述。图10为用长度为96bp的环状双链DNA模板制备双链RNA的结果。结果显示转录产物也为双链RNA，表明本方法具有良好的通用性。
实施例8
(1)原料
A链
Figure PCTCN2022092023-appb-000086
CCAGGAATCAGCGGCAAATT CCTCTACTTTCCTCGTCACATCTT(
Figure PCTCN2022092023-appb-000087
-磷酸化，长度为44nt，SEQ ID NO：14，下划线处为泡状结构所在位置)；
B链
Figure PCTCN2022092023-appb-000088
CGACTCCTGTACTGACAACACTCTCACACGACACATCCGCGTCACAAAAGGT(
Figure PCTCN2022092023-appb-000089
-磷酸化，长度为52nt，SEQ ID NO：15)；
C链
Figure PCTCN2022092023-appb-000090
GAAGAAACTCATGACATCGAATTGTTGAGCGAAGAATATGACGCCACTCCTTTCATCAA(
Figure PCTCN2022092023-appb-000091
-磷酸化，长度为59nt，SEQ ID NO：22)；
Splint AB
Figure PCTCN2022092023-appb-000092
ACAGGAGTCGAAGATGTGAC(长度为20nt，SEQ ID NO：16)；
Splint BC
Figure PCTCN2022092023-appb-000093
GAGTTTCTTCACCTTTTGTG(长度为20nt，SEQ ID NO：23)；
Splint CA
Figure PCTCN2022092023-appb-000094
TGATTCCTGGTTGATGAAAG(长度为20nt，SEQ ID NO：24)；
A1链
Figure PCTCN2022092023-appb-000095
AAGATGTGA GCTCCTTTCATCTCCAATTTGCCGCTGATTCCTGG(5-磷酸化，长度为44nt， SEQ ID NO：18，下划线处为泡状结构所在位置)；
B1链
Figure PCTCN2022092023-appb-000096
ACCTTTTGTGACGCGGATGTGTCGTGTGAGAGTGTTGTCAGTACAGGAGTCG(5-磷酸化，长度为52nt，SEQ ID NO：19)；
C1链
Figure PCTCN2022092023-appb-000097
TTGATGAAAGGAGTGGCGTCATATTCTTCGCTCAACAATTCGATGTCATGAGTTTCTTC(5-磷酸化，长度为59nt，SEQ ID NO：25)；
Splint A1C1(5→3)：
CTTTCATCAACCAGGAATCA(长度为20nt，SEQ ID NO：26)；
Splint C1B1
Figure PCTCN2022092023-appb-000098
CACAAAAGGTGAAGAAACTC(长度为20nt，SEQ ID NO：27)；
Splint B1A1
Figure PCTCN2022092023-appb-000099
CTCACATCTTCGACTCCTGT(长度为20nt，SEQ ID NO：21)。
155nt的单环制备方法不同于实施例1中的一锅法，采用分步的方式制备。具体地，先制备L-A1B1，再以L-A1B1和C1为原料制备C-A1B1C1(155nt的单环)。
L-A1B1的制备：
将A1和B1(终浓度为4μM)、splint(Splint B1A1，终浓度为6μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.25U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为1×)混合，37℃下反应2h。
C-A1B1C1的制备：
将制备好的L-A1B1(终浓度为2μM)、C1(终浓度为2μM)、splint(Splint A1C1和Splint C1B1，终浓度均为3μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.125U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为0.5×)混合，37℃下反应2h。
155bp的环状双链DNA模板的制备方法与实施例7相同。具体地：将制备好的155nt单环和与其互补链对应的三条短DNA链(A、B和C)退火杂交(退火条件同实施例1)后直接加入Taq Dnl进行连接。155bp的环状双链DNA模板的纯化方法及后续的转录条件同实施例1，此处不再赘述。
图11为用长度为155bp的环状双链DNA模板制备双链RNA的结果。结果显示转录产物也为双链RNA，表明本方法具有良好的通用性。
实施例9
(1)原料
B链
Figure PCTCN2022092023-appb-000100
CGACTCCTGTACTGACAACACTCTCACACGACACATCCGCGTCACAAAAGGT(
Figure PCTCN2022092023-appb-000101
-磷酸化，长度为52nt，SEQ ID NO：15)；
C链
Figure PCTCN2022092023-appb-000102
GAAGAAACTCATGACATCGAATTGTTGAGCGAAGAATATGACGCCACTCCTTTCATCAA(
Figure PCTCN2022092023-appb-000103
-磷酸化，长度为59nt，SEQ ID NO：22)；
D链
Figure PCTCN2022092023-appb-000104
AATCGGCAGTCCATT CGCAGAAGCAACTGTACTCAAATTCGGTAAACTCCAACGC(
Figure PCTCN2022092023-appb-000105
-磷酸化，长度为55nt，SEQ ID NO：28，下划线处为泡状结构所在位置)；
E链
Figure PCTCN2022092023-appb-000106
ACTCAGTACGCATACTTCGTCACTGCTGATGACATCAGGGTTGGTTCAATGTCCGCCGA(
Figure PCTCN2022092023-appb-000107
-磷酸化，长度为59nt，SEQ ID NO：29)；
F链
Figure PCTCN2022092023-appb-000108
CGGCTACCACAACATTTCTACCAAGGATGGTGACTGCGGTTCACTCCTCTTTG(
Figure PCTCN2022092023-appb-000109
-磷酸化，长度为53nt，SEQ ID NO：30)；
Splint BC
Figure PCTCN2022092023-appb-000110
GAGTTTCTTCACCTTTTGTG(长度为20nt，SEQ ID NO：23)；
Splint CD
Figure PCTCN2022092023-appb-000111
ACTGCCGATTTTGATGAAAG(长度为20nt，SEQ ID NO：31)；
Splint DE
Figure PCTCN2022092023-appb-000112
CGTACTGAGTGCGTTGGAGT(长度为20nt，SEQ ID NO：32)；
Splint EF
Figure PCTCN2022092023-appb-000113
GTGGTAGCCGTCGGCGGACA(长度为20nt，SEQ ID NO：33)；
Splint FB
Figure PCTCN2022092023-appb-000114
ACAGGAGTCGCAAAGAGGAG(长度为20nt，SEQ ID NO：34)；
B1链
Figure PCTCN2022092023-appb-000115
ACCTTTTGTGACGCGGATGTGTCGTGTGAGAGTGTTGTCAGTACAGGAGTCG(
Figure PCTCN2022092023-appb-000116
-磷酸化，长度为52nt，SEQ ID NO：19)；
C1链
Figure PCTCN2022092023-appb-000117
TTGATGAAAGGAGTGGCGTCATATTCTTCGCTCAACAATTCGATGTCATGAGTTTCTTC (
Figure PCTCN2022092023-appb-000118
-磷酸化，长度为59nt，SEQ ID NO：25)；
D1链
Figure PCTCN2022092023-appb-000119
GCGTTGGAGTTTACCGAATTTGAGT TGTCAACGAAGACGCAATGGACTGCCGATT
(
Figure PCTCN2022092023-appb-000120
-磷酸化，长度为55nt，SEQ ID NO：35，下划线处为泡状结构所在位置)；
E1链
Figure PCTCN2022092023-appb-000121
TCGGCGGACATTGAACCAACCCTGATGTCATCAGCAGTGACGAAGTATGCGTACTGAGT(
Figure PCTCN2022092023-appb-000122
-磷酸化，长度为59nt，SEQ ID NO：36)；
F1链
Figure PCTCN2022092023-appb-000123
CAAAGAGGAGTGAACCGCAGTCACCATCCTTGGTAGAAATGTTGTGGTAGCCG(
Figure PCTCN2022092023-appb-000124
-磷酸化，长度为53nt，SEQ ID NO：37)；
Splint C1B1
Figure PCTCN2022092023-appb-000125
CACAAAAGGTGAAGAAACTC(长度为20nt，SEQ ID NO：27)；
Splint D1C1
Figure PCTCN2022092023-appb-000126
CTTTCATCAAAATCGGCAGT(长度为20nt，SEQ ID NO：38)；
Splint E1D1
Figure PCTCN2022092023-appb-000127
ACTCCAACGCACTCAGTACG(长度为20nt，SEQ ID NO：39)；
Splint F1E1
Figure PCTCN2022092023-appb-000128
TGTCCGCCGACGGCTACCAC(长度为20nt，SEQ ID NO：40)；
Splint B1F1
Figure PCTCN2022092023-appb-000129
CTCCTCTTTGCGACTCCTGT(长度为20nt，SEQ ID NO：41)。
278nt的单环制备方法同实施例8，即采用分步的方式制备。具体地，先制备L-B1C1和L-D1E1，再以L-D1E1和F1为原料制备L-D1E1F1，最后以L-B1C1和L-D1E1F1为原料制备C-B1C1D1E1F1(278nt的单环)。
L-B1C1的制备：
将B1和C1(终浓度为4μM)、splint(Splint C1B1，终浓度为6μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.25U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为1×)混合，37℃下反应2h。
L-D1E1的制备：
将D1和E1(终浓度为4μM)、splint(Splint E1D1，终浓度为6μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.25U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为1×)混合，37℃下反应2h。
L-D1E1F1的制备：
将制备好的L-D1E1(终浓度为2μM)、F1(终浓度为2μM)、splint(Splint F1E1，终浓度为3μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.125U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为0.5×)混合，37℃下反应2h。
C-B1C1D1E1F1的制备：
将制备好的L-D1E1F1(终浓度为1μM)和L-B1C1(终浓度为1μM)、splint(Splint D1C1和Splint B1F1，终浓度均为1.5μM)、T4 DNA连接酶(T4 Dnl，终浓度为0.0625U/μL)和对应的T4 DNA连接酶缓冲液(终浓度为0.25×)混合，37℃下反应2h。
278bp的环状双链DNA模板的制备方法与实施例7和实施例8相同。具体地：将制备好的278nt单环和与其互补链对应的五条短DNA链(B、C、D、E和F)退火杂交(退火条件同实施例1)后直接加入Taq Dnl进行连接。278bp的环状双链DNA模板的纯化方法及后续的转录条件同实施例1，此处不再赘述。图12为用长度为278bp的环状双链DNA模板制备双链RNA的结果。结果显示转录产物也为双链RNA，表明本方法具有良好的通用性。
实施例10
所用的原料，单环、单链及环状双链DNA的制备及纯化方法同实施例1，此处不再赘述。不同之处在于这里用SP6 RNA聚合酶进行转录，转录条件同实施例1。
图13为B15在SP6 RNA聚合酶的作用下制备双链RNA的结果。结果显示转录产物也为双链RNA，表明本方法具有良好的通用性，适用于多种常见的RNA聚合酶。
以上所述的实施例仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (10)

  1. 一种制备双链RNA的方法，其特征在于，包括以下步骤：
    步骤一，设计并制备环状双链DNA模板，所述环状双链DNA模板含有非互补的泡状结构部分和互补部分，不含启动子序列，所述互补部分与要制备的双链RNA长度相同且序列相同，序列中T对应U，所述泡状结构处于环状双链DNA模板中互补部分的两端，泡状结构的长度为5-15bp，泡状结构的两条链之间连续互补的碱基不超过2个；
    步骤二，将制备的环状双链DNA模板纯化，去除环状双链DNA模板中混杂着的线性单链DNA；
    步骤三，将纯化的环状双链DNA模板、RNA聚合酶、核糖核酸酶H、NTPs、RNA酶抑制剂、无机焦磷酸酶和相应的RNA聚合酶缓冲液混合，进行转录，所述RNA聚合酶为T7 RNA聚合酶、SP6 RNA聚合酶或者T3 RNA聚合酶，在转录过程中RNA聚合酶进行双向滚环转录，核糖核酸酶H则在环状双链DNA模板中泡状结构处DNA的辅助下对含有重复序列的转录产物进行酶切，获得目的双链RNA。
  2. 根据权利要求1所述的制备双链RNA的方法，其特征在于，所述泡状结构的长度为5-15bp。
  3. 根据权利要求1所述的制备双链RNA的方法，其特征在于，所述双链RNA的长度为70-1000bp。
  4. 根据权利要求1所述的制备双链RNA的方法，其特征在于，所述双链RNA的长度为96-278bp。
  5. 根据权利要求1-4任一项所述的制备双链RNA的方法，其特征在于，当所述环状双链DNA模板长度小于90bp时，采用一段法，设计一条成环前体线性DNA链制备环状单链DNA，环状单链DNA和线性单链DNA退火杂交后加入DNA连接酶和对应的连接酶缓冲液进行连接反应；
    当所述环状双链DNA模板长度大于90bp时，采用一锅法或分步法制备环状单链DNA，然后将环状单链DNA和其互补链所对应的多条短DNA链混合，退火杂交后加入DNA连接酶和对应的连接酶缓冲液进行连接反应；
    所述环状单链DNA和线性单链DNA杂交后，线性单链DNA的两端距离泡状结构的边缘大于15nt。
  6. 根据权利要求5所述的制备双链RNA的方法，其特征在于，所述退火杂交条件为：先在90℃下保温1-3min，然后以0.1℃/s的速度降温至20-25℃并保温10min。
  7. 根据权利要求5所述的制备双链RNA的方法，其特征在于，当使用T4DNA连接酶和相 应的T4DNA连接酶缓冲液时，在4-37℃温度范围内保温2-24h进行连接；当使用TaqDNA连接酶和相应的TaqDNA连接酶缓冲液时，在45-80℃范围内保温2-24h进行连接。
  8. 根据权利要求7所述的制备双链RNA的方法，其特征在于，当使用T4DNA连接酶时，采用终浓度为0.05-1×的缓冲液，当缓冲液浓度为1×时，其组成为：40mM Tris-HCl，10mM MgCl 2，10mM DTT，500μM ATP，pH 7.8@25℃；当使用TaqDNA连接酶时，采用终浓度为1×的缓冲液，其组成为：20mM Tris-HCl，25mM KAc，10mM Mg(Ac) 2，10mM DTT，1.0mM NAD，0.1％Triton X-100，pH 7.6@25℃。
  9. 根据权利要求1所述的制备双链RNA的方法，其特征在于，所述步骤二的双链环状DNA模板的纯化过程中，用ExoI和ExoIII共同酶切去除环状双链DNA模板中混杂着的线性单链DNA，Exo I的酶切反应采用终浓度为0.5×的缓冲液，其组成为：33.5mM glycine-KOH，pH 9.5@25℃，3.35mM MgCl 2，0.5mM DTT；Exo III的酶切反应采用终浓度为0.5×的缓冲液，其组成为：33mM Tris-HCl，pH 8.0@30℃；0.33mM MgCl 2。
  10. 根据权利要求1所述的制备双链RNA的方法，其特征在于，所述步骤三在1×RNA聚合酶缓冲液中进行，成分为：40mM Tris-HCl，pH 7.9@25℃，6.0mM MgCl 2，10mM DTT，10mM NaCl和2mM spermidine，在37℃下恒温反应0.5-72h制备双链RNA。
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