WO2022170705A1 - 一种制备定点修饰的长链rna的方法 - Google Patents

一种制备定点修饰的长链rna的方法 Download PDF

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WO2022170705A1
WO2022170705A1 PCT/CN2021/098422 CN2021098422W WO2022170705A1 WO 2022170705 A1 WO2022170705 A1 WO 2022170705A1 CN 2021098422 W CN2021098422 W CN 2021098422W WO 2022170705 A1 WO2022170705 A1 WO 2022170705A1
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nucleic acid
rna
strand
fragment
acid fragment
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French (fr)
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刘冬生
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清华大学
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • the present disclosure belongs to the field of molecular biology and synthetic biology, and in particular, the present disclosure relates to a method for preparing long-chain RNA and the prepared long-chain RNA.
  • RNA-based immunotherapy has gradually shown remarkable interest in the fields of biomedicine such as inherited metabolic diseases [1,2] , tumor therapy [3-5] , antiviral therapy [6,7] and regenerative medicine.
  • RNA technologies such as siRNA, miRNA, long non-coding RNA (Lnc RNA), RNA aptamers and ribozymes are also developing rapidly, and RNA has received more and more attention in biology.
  • Lnc RNA long non-coding RNA
  • RNA aptamers and ribozymes are also developing rapidly, and RNA has received more and more attention in biology.
  • RNA has shown rich application prospects in the field of biomedicine.
  • its low stability in biological systems greatly limits the practical therapeutic applications of unmodified RNAs.
  • chemical modifications such as base modification [8] , ribose modification [9] and phosphate backbone modification [10] , etc.
  • RNA synthesis methods include solid-phase synthesis, in vitro transcription, rolling circle transcription (RCT) and other methods.
  • the solid-phase synthesis method (phosphoramidite method) is the most widely used synthesis method at present, which can usually synthesize short-chain RNAs of 60 nt and below [12] on a large scale, and is suitable for precise modification of any site in RNA.
  • the solid-phase synthesis method is used to synthesize target RNAs with longer sequence lengths, the synthetic yield decreases exponentially with the increase of sequence length, and the error rate increases rapidly.
  • RNA sequences synthesized by chemical synthesis with a certain yield are still generally 60nt in length [12] ; the maximum length of commercial single-stranded RNA synthesis can reach 120nt, but for RNA sequences containing site-directed modifications within the sequence (that is, on the bases other than the two bases at the 5' and 3' ends) can only reach 60nt at most.
  • RNAs For longer RNAs, their preparation is currently mainly accomplished by in vitro transcription.
  • T7, T3 or SP6 RNA polymerase the double-stranded DNA template with promoter can be transcribed to obtain long single-stranded RNA, a large amount of RNA products can be obtained, and the transcription length is not limited, which is suitable for the synthesis of long-stranded RNA [14] .
  • Bieker et al. [15] first realized the transcription of 5S DNA based on RNA polymerase III in 1984.
  • the in vitro transcription method is highly dependent on RNA polymerase, and the insertion of modified bases in the sequence can only be achieved by means of enzyme engineering [16] and adding a certain proportion of modified base monomers [8] .
  • RNA sequence of modified bases is a mixture of multiple sequences at the molecular level, which hinders the in-depth study of site-specific chemical modification for its application in biomedicine.
  • the RCT method can also realize the synthesis of long-chain RNA, but this method is generally used for the synthesis of continuous repeating sequences [17] , its application scope is more limited, and there is also the problem that the site-directed insertion of modified bases in the sequence cannot be achieved [18] . Therefore, there is an urgent need to develop new RNA synthesis methods that can achieve stable and large-scale production of long-chain RNAs and meet the needs of precise insertion of specific modified bases in RNAs.
  • the first step is to design a double-stranded DNA transcription template; the second Step 3, template preparation; Step 3, template purification; Step 4, in vitro transcription; Step 5, purification of RNA product; Step 6, detection of RNA purity after purification.
  • the above-mentioned technology has realized large-scale synthesis of long-chain RNA drugs, it still belongs to the traditional molecular biology process, namely: based on DNA template, utilize RNA polymerase to transcribe in vitro, and obtain 30-200nt RNA single strand after purification.
  • the method described in this patent does not involve the preparation of RNA containing unnatural modified bases, and from the principles described in the preceding paragraph, it can be known that this method cannot achieve the preparation of long RNA chains containing modified bases at specific sites.
  • the present disclosure provides a method for preparing long-chain RNA, which is capable of synthesizing long-chain RNAs with arbitrary sequences in the range of 60 nt or more, especially in the range of 60-1000 nt, based on the precise assembly and ligation process.
  • the method of the present disclosure can be used to prepare long-chain RNAs that can be precisely modified at any site, overcoming the current technical obstacle of inability to synthesize long-chain RNAs with precise site modifications.
  • the method of the present disclosure can be used to prepare single-stranded long-stranded RNA, and the prepared single-stranded long-stranded RNA can contain a modification group at any position, and has the advantages of low synthesis difficulty, high accuracy, low cost advantage.
  • the present disclosure provides a method for preparing long-chain RNA, comprising the following steps:
  • Synthesis step Synthesize the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand, the nucleic acid fragment group of the first strand is composed of RNA fragments, and the nucleic acid fragment group of the second strand is composed of RNA fragments and DNA fragments. at least one of the constituents;
  • the nucleic acid fragment group of the first strand includes an RNA fragment group a and an optional RNA fragment group b
  • the nucleic acid fragment group of the second strand includes a nucleic acid fragment group c and an optional nucleic acid fragment group d
  • the RNA fragment Group a includes RNA fragments n i and RNA fragments n i+1
  • said nucleic acid fragment group b includes RNA fragments x ii and RNA fragments x ii+1
  • said nucleic acid fragment group c includes nucleic acid fragments mi
  • said nucleic acid fragments Group d includes at least one of nucleic acid fragments y ii and nucleic acid fragments y ii+1 , where i and ii are independently selected from integers of 1 or more;
  • the 5'-end sequence of the nucleic acid fragment mi and the 5'-end sequence of the RNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the RNA fragment n i are complementary sequences
  • Annealing step mixing the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand in the same reaction system and annealing to form a double-stranded assembly precursor; There is a junction between each nucleic acid fragment, and a junction exists between two adjacent nucleic acid fragments in the second strand; the junction between the adjacent nucleic acid fragments in the nucleic acid fragment group of the first strand is the same as the The connection ports between adjacent nucleic acid fragments in the nucleic acid fragment group of the second strand are staggered from each other;
  • Connecting step connecting the connecting port of the first strand to obtain a double-stranded assembly formed by the complementation of the continuous single-stranded RNA and the fragmented single-stranded nucleic acid strand.
  • the set of nucleic acid fragments of the second strand consists of RNA fragments.
  • the set of nucleic acid fragments of the second strand consists of DNA fragments.
  • the group of nucleic acid fragments of the second strand consists of RNA fragments and DNA fragments.
  • the method for preparing long-chain RNA according to the present disclosure wherein the method further comprises the steps of:
  • Denaturation step denaturing the double-stranded assembly to obtain a continuous single-stranded RNA
  • the method further includes a purification step: purifying the continuous single-stranded RNA from the reaction system.
  • the hairpin structure further comprises a stem region that forms a double-stranded structure and a stem-loop region that does not form a double-stranded structure
  • the stem The 5' end and the 3' end of the stem region are respectively connected to the 5' extension arm and the 3' extension arm; preferably, the junction between the RNA fragment x ii and RNA fragment x ii+1 is located in the stem area.
  • the 3'-end sequence of the RNA fragment n i+1 and the 3'-end sequence of other nucleic acid fragments of the nucleic acid fragment group c are complementary sequences or unpaired sequences; or,
  • the 3'-end sequence of the nucleic acid fragment y ii and the 3'-end sequence of the other nucleic acid fragments of the RNA fragment group a are complementary sequences or unpaired sequences; or,
  • the 5'-end sequence of the nucleic acid fragment y ii+1 and the 5'-end sequence of other nucleic acid fragments of the RNA fragment group a are complementary sequences or unpaired sequences;
  • the 3'-end sequence of the RNA fragment n i+1 and the 3'-end sequence of the nucleic acid fragment m i+1 are complementary sequences, and the 5'-end sequence of the nucleic acid fragment m i+1 is the same as the nucleic acid fragment mi+1 .
  • the other nucleic acid fragments of fragment group a are complementary sequences or unpaired sequences.
  • the length of the continuous single-stranded RNA is 60nt or more, preferably 80nt or more, preferably 100nt or more, preferably 120nt or more, preferably 60- 1000nt, preferably 80-600nt, more preferably 100-400nt, most preferably 120-360nt.
  • the method for preparing long-chain RNA according to the present disclosure wherein the length of any nucleic acid fragment in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 8- 120nt, preferably 10-80nt, more preferably 15-40nt, most preferably 20-30nt.
  • the method for preparing long-chain RNA according to the present disclosure wherein the 5'-end sequence of any nucleic acid fragment in the first-strand nucleic acid fragment group and the second-strand nucleic acid fragment group
  • the length is 4nt or more, preferably 4-50nt, more preferably 6-30nt, most preferably 10-20nt; or,
  • the length of the 3' end sequence of any nucleic acid fragment in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 4 nt or more, preferably 4-50 nt, more preferably 6-30 nt, most preferably 10- 20nt.
  • the method for preparing long-chain RNA according to the present disclosure wherein any nucleic acid fragment in the set of nucleic acid fragments of the first strand comprises a phosphate group at the 5' end, and a phosphate group at the 3' end
  • the phosphoric acid groups and hydroxyl groups on both sides of the connecting port are connected as phosphodiester bonds;
  • the adjacent phosphate groups and hydroxyl groups are linked as phosphodiester linkages by enzymatic or chemical linkages.
  • the method for preparing long-chain RNA according to the present disclosure wherein one or more of any one of the nucleic acid fragments in the first-strand nucleic acid fragment group and the second-strand nucleic acid fragment group comprising a modified base at the position, and the base at the position immediately adjacent to the junction is an unmodified base;
  • the modification is selected from m 6 A, ⁇ , m 1 A, m 5 A, ms 2 i 6 A, i 6 A, m 3 C, m 5 C, ac 4 C, m 7 G, m 2 ,2G, m2G , m1G , Q, m5U , mcm5U , ncm5U , ncm5Um , D, mcm5s2U , Inosine (I), hm5C , s4U , s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or derivatives thereof.
  • the method for preparing long-chain RNA according to the present disclosure wherein one or more of any one of the nucleic acid fragments in the first-strand nucleic acid fragment group and the second-strand nucleic acid fragment group comprising a modified ribose at the position, and the ribose at the position immediately adjacent to the junction is an unmodified ribose;
  • the modification is selected from LNA, 2'-OMe, 3'-OMeU, vmoe, 2'-F or 2'-OBn (2'-O-benzyl group) or derivatives thereof.
  • any nucleic acid fragment in the set of nucleic acid fragments of the first strand comprise modified phosphodiester bonds, And the phosphodiester bond at the position adjacent to the connection port is an unmodified phosphodiester bond;
  • one or more positions of any nucleic acid fragment in the nucleic acid fragment group of the second strand comprise a modified phosphodiester bond, and the phosphodiester bond at the position immediately adjacent to the connecting port is an unmodified phosphate diester bond;
  • the modification is selected from phosphorothioate (PS), nucleotide triphosphate (NTP ⁇ S) or derivatives thereof, and the like.
  • the temperature of the incubation is any temperature of 0-100°C, preferably any temperature of 10-85°C, more preferably any temperature in the interval of 20-65°C, and the incubation time is any desired time;
  • the speed of cooling can be any speed, and the temperature can be lowered to any temperature at which the nucleic acid fragments in the reaction system are hybridized to form the precursor of the double-stranded assembly.
  • the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand are dissolved in the same solvent to obtain the reaction system.
  • the pH of the reaction system is 3-11, preferably pH 4-10, more preferably pH 5-9, most preferably pH 6- 8.
  • any two nucleic acid fragments in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 1:(0.1-10), preferably 1:(0.5-2), most preferably 1:1.
  • the present disclosure also provides a long-chain RNA, wherein the long-chain RNA is prepared by the method according to the present disclosure, and the long-chain RNA is a single-stranded long-chain RNA;
  • the long RNA comprises modified base, ribose or phosphodiester linkages at one or more positions.
  • the method for preparing long-chain RNA provided by the present disclosure can prepare long-chain RNA of any sequence, and the method described in the present invention does not require DNA as a template, does not rely on RNA polymerase, etc., and has a cost It has the advantages of low synthesis, low synthesis difficulty, high yield, high sequence accuracy, and precise introduction of modifications, making it suitable for large-scale production applications.
  • the method for preparing long-chain RNA provided by the present disclosure can prepare long-chain RNA with a hairpin structure to simulate the natural spatial conformation of RNA molecules, and on the one hand, improves the structural stability of long-chain RNA, On the other hand, it is beneficial to realize the biological function of long-chain RNA.
  • the method for preparing long-chain RNA provided by the present disclosure, by preparing a double-stranded assembly formed by the complementarity of a continuous single-stranded RNA and a fragmented single-stranded nucleic acid strand, the double-stranded assembly only needs to be simply
  • the target long-stranded RNA can be obtained by the denaturation treatment, and the non-target single-stranded nucleic acid strands are dispersed in the reaction system as nucleic acid fragments after the preparation is completed, and there is no need for reprocessing such as shearing or effect, which effectively simplifies
  • the preparation steps of single-stranded long-chain RNA are improved, and the preparation efficiency is improved.
  • the method for preparing long-chain RNA provided by the present disclosure in which precise insertion of modified bases at any site is achieved, solves the problem that the current synthesis methods for long-chain RNA cannot achieve precise modification of specific sites. question.
  • the long-chain RNA provided by the present disclosure is prepared by the above-mentioned method for preparing long-chain RNA, the sequence accuracy is high, and the precise insertion of modified bases at any site can be realized. and long-chain RNAs containing modified bases, which have broad application prospects in drug development and clinical treatment.
  • Fig. 1 shows the assembly schematic diagram of long-chain RNA
  • Figure 2 shows a schematic diagram of the assembly of a hairpin structure in a long RNA
  • Figure 3 shows a schematic diagram of the assembly of long RNAs comprising hairpin structures
  • Figure 4 shows the results of native polyacrylamide gel electrophoresis characterization of RNA80/100 assemblies
  • Figure 5 shows the results of denaturing polyacrylamide gel electrophoresis characterization of 80nt RNA single strands
  • Figure 6 shows the characterization results of native polyacrylamide gel electrophoresis of three double-stranded RNA assemblies of RNA100/100, RNA140/120 and RNA200/180;
  • Figure 7 shows the characterization results of denaturing polyacrylamide gel electrophoresis of 100nt, 140nt and 200nt RNA single strands
  • Figure 8 shows the results of denaturing polyacrylamide gel electrophoresis characterization of single strands of RNA up to 267 nt;
  • Figure 9 shows the characterization results of denaturing polyacrylamide gel electrophoresis of 267nt RNA single strands containing site-directed modifications.
  • numerical range represented by "numerical value A to numerical value B" refers to the range including the numerical values A and B at the endpoints.
  • multiple in “multiple”, “plurality”, “plurality”, etc. means a numerical value of 2 or more.
  • the "substantially”, “substantially” or “substantially” means that the error is less than 5%, or less than 3% or less than 1% compared to the relevant perfect standard or theoretical standard.
  • water includes tap water, deionized water, distilled water, double-distilled water, purified water, ion-exchanged water, and the like, any practicable water.
  • double-stranded RNA assembly and “double-stranded RNA” have the same meaning and can be substituted for each other.
  • double-stranded assembly and “double-stranded assembly precursor” may be formed by the complementarity of continuous single-stranded RNA and fragmented single-stranded RNA, or may be formed by continuous single-stranded RNA and fragmented single-stranded RNA
  • the formation of complementary single-stranded DNA can also be formed by the complementation of continuous single-stranded RNA and fragmented single-stranded nucleic acid formed by RNA fragments and DNA fragments. That is, in the present disclosure, the double strands in the "double-stranded assembly” and the “double-stranded assembly precursor" are not DNA double strands.
  • connection port is also called a nick, which exists between two adjacent nucleotides of a single-stranded nucleic acid chain because no phosphate is formed between the adjacent two nucleotides. produced by the diester bond.
  • a first aspect of the present disclosure provides a method for preparing long-chain RNA, comprising the steps of:
  • Synthesis step Synthesize the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand, the nucleic acid fragment group of the first strand is composed of RNA fragments, and the nucleic acid fragment group of the second strand is composed of RNA fragments and DNA fragments. at least one of the constituents;
  • the nucleic acid fragment group of the first strand includes an RNA fragment group a and an optional RNA fragment group b
  • the nucleic acid fragment group of the second strand includes a nucleic acid fragment group c and an optional nucleic acid fragment group d
  • the RNA fragment Group a includes RNA fragments n i and RNA fragments n i+1
  • said nucleic acid fragment group b includes RNA fragments x ii and RNA fragments x ii+1
  • said nucleic acid fragment group c includes nucleic acid fragments mi
  • said nucleic acid fragments Group d includes at least one of nucleic acid fragments y ii and nucleic acid fragments y ii+1 , where i and ii are independently selected from integers of 1 or more;
  • the 5'-end sequence of the nucleic acid fragment mi and the 5'-end sequence of the RNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the RNA fragment n i are complementary sequences
  • Annealing step mixing the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand in the same reaction system and annealing to form a double-stranded assembly precursor; There is a junction between each nucleic acid fragment, and a junction exists between two adjacent nucleic acid fragments in the second strand; the junction between the adjacent nucleic acid fragments in the nucleic acid fragment group of the first strand is the same as the The connection ports between adjacent nucleic acid fragments in the nucleic acid fragment group of the second strand are staggered from each other;
  • Connecting step connecting the connecting port of the first strand to obtain a double-stranded assembly formed by the complementation of the continuous single-stranded RNA and the fragmented single-stranded nucleic acid strand.
  • Patent document CN102876658A discloses a method for large-scale synthesis of long-chain nucleic acid molecules.
  • the small fragment nucleic acid molecules synthesized in step 1 and comprising the above-mentioned purified sequence are added in solution by complementary Pairing to form a double-strand with a gap in each single-strand, under the action of nucleic acid molecule ligase, the gaps on the two strands are connected to form a long double-strand assembly, and a large number of mixed products are processed through the PCR step. Amplify and purify the long double-stranded assembly in the final product, and finally obtain a long double-stranded nucleic acid molecule formed by the complementation of two continuous single strands.
  • the present disclosure found in research that the above-mentioned preparation method of long double-stranded nucleic acid molecule has at least the following problems: (1) the nucleic acid molecule with a double-stranded structure formed by two continuous long chains is obtained by the method disclosed above, After the denaturation of the double-stranded structure, two continuous long chains are uniformly mixed in the reaction system. Therefore, when a single-stranded long-chain nucleic acid molecule needs to be prepared, it cannot be achieved only by melting the double-stranded structure. In fact, for double-stranded molecules up to 60bp, the melting temperature is extremely high, and the melting process is difficult.
  • RNA double strands cannot be directly amplified by PCR, so the above disclosed method cannot be directly used for RNA preparation.
  • the amplification of a small amount of target long double-stranded nucleic acid molecules in the mixed crude product must be achieved through the PCR process, and the introduction of precise site-specific modification into the final product cannot be achieved through the PCR process.
  • the preparation method of the present disclosure divides the long-chain RNA into several short RNA fragments, which greatly reduces the difficulty of synthesizing the long-chain RNA.
  • it is not necessary to use DNA as a template or use RNA polymerase. It is difficult to chemically synthesize, and it can modify the base, ribose or phosphodiester bond at any position in the long-chain RNA, avoiding the problem that the conventional in vitro transcription method is difficult to achieve site-directed modification, and has the advantages of low cost, high yield, sequence The advantage of high accuracy.
  • the preparation method of the present disclosure only connects the junction of the first strand formed by the RNA fragment to obtain a double-stranded assembly formed by the complementarity of the continuous single-stranded RNA and the fragmented single-stranded nucleic acid strand.
  • the single-stranded nucleic acid strand may be composed of at least one of DNA fragments and RNA fragments.
  • the preparation method also does not include an amplification step, and avoids connecting the junction ports of the fragmented single-stranded nucleic acid strands (that is, single-stranded RNA) in the double-stranded assembly, which is only obtained by simple The denaturation treatment can realize the recovery of the target long-chain RNA.
  • the preparation method does not include steps such as digestion and shearing of the non-target single-stranded nucleic acid chain, which effectively improves the preparation efficiency of the single-stranded long-chain RNA. It is suitable for large-scale industrial applications.
  • Figure 1 shows a long-chain double-stranded RNA structure, wherein the first strand is the target synthesized long-chain RNA, and the second strand is the target synthetic long-chain RNA.
  • the nucleotide sequences of the first strand and the second strand are respectively divided, so that the nucleotide sequences of the first strand and the second strand are divided into several short-chain nucleic acid fragment sequences.
  • the group of nucleic acid fragments forming the first strand is composed of RNA fragments
  • the group of nucleic acid fragments forming the second strand is composed of at least one of RNA fragments and DNA fragments.
  • nucleic acid fragment group of the first strand is selected from RNA fragment group a and optional RNA fragment group b
  • nucleic acid fragment group of the second strand is selected from nucleic acid fragment group c and optional nucleic acid fragment group d.
  • nucleic acid fragment group of the first strand it can be RNA fragment group a, or a combination of RNA fragment group a and RNA fragment group b; for the nucleic acid fragment group of the second strand, it can be nucleic acid fragment group c, or nucleic acid fragment group Combination of group c and nucleic acid fragment group d.
  • nucleic acid fragment group c consists of RNA fragments and nucleic acid fragment group d consists of RNA fragments. In some embodiments, nucleic acid fragment group c consists of DNA fragments and nucleic acid fragment group d consists of DNA fragments. In some embodiments, nucleic acid fragment group c consists of RNA fragments and DNA fragments, and nucleic acid fragment group d consists of RNA fragments and DNA fragments. In some embodiments, nucleic acid fragment group c consists of RNA fragments and DNA fragments, and nucleic acid fragment group d consists of RNA fragments.
  • nucleic acid fragment group c consists of RNA fragments and nucleic acid fragment group d consists of RNA fragments and DNA fragments. In some embodiments, nucleic acid fragment group c consists of RNA fragments and DNA fragments, and nucleic acid fragment group d consists of DNA fragments. In some embodiments, nucleic acid fragment group c consists of DNA fragments and nucleic acid fragment group d consists of RNA fragments and DNA fragments.
  • RNA fragment group a includes RNA fragment n i and RNA fragment n i+1
  • nucleic acid fragment group c includes nucleic acid fragment m i , the 5' end sequence of nucleic acid fragment mi and the 5' end sequence of RNA fragment n i+1
  • the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the RNA fragment ni are complementary sequences.
  • RNA fragment group b includes RNA fragment x ii and RNA fragment x ii+1
  • nucleic acid fragment group d includes nucleic acid fragment y ii , nucleic acid fragment y ii+1 or both nucleic acid fragment y ii and nucleic acid fragment y ii+1 .
  • RNA fragment x ii and RNA fragment x ii+1 are complementary to form a hairpin structure with a 5' extension arm and a 3' extension arm, the 5' extension arm sequence and the 5' end of the nucleic acid fragment y ii
  • the sequence is a complementary sequence
  • the 3' extension arm sequence and the 3' end sequence of the nucleic acid fragment y ii+1 are complementary sequences.
  • i and ii are independently selected from an integer of 1 or more.
  • the RNA fragment group of the first strand is RNA fragment group a
  • the RNA fragment group of the second strand is RNA fragment group c, that is, the RNA sequence of the first strand is divided into the sequence of RNA fragment n i and The sequence of the RNA fragment n i+1 , the nucleic acid sequence of the second strand complementary to the first strand is divided into the sequence of the nucleic acid fragment mi, formed by the sequence of the 5' end of the nucleic acid fragment mi and the sequence of the nucleic acid fragment n i +1 .
  • the complementary pairing of the 5'-end sequence, the complementary pairing of the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the nucleic acid fragment ni realizes the sequence division of the double-stranded RNA comprising the target long-stranded RNA (first strand) .
  • the nucleic acid fragment mi can be a DNA fragment or an RNA fragment, both of which can realize the sequence synthesis of the target long-chain RNA.
  • RNA fragment group a may also contain other nucleic acid fragments.
  • RNA fragment group a includes RNA fragment n i , RNA fragment n i+1 , RNA fragment n i+2 .
  • RNA fragment set a includes RNA fragment n i , RNA fragment n i+1 , RNA fragment n i+2 , RNA fragment n i+3 .
  • RNA fragment group a includes RNA fragment n i , RNA fragment n i+1 , RNA fragment n i+2 , RNA fragment n i+3 , RNA fragment n i+4 .
  • the RNA fragment group a may also include other numbers of RNA fragments, which are not exhaustive in the present disclosure.
  • nucleic acid fragment group c may also include other nucleic acid fragments.
  • nucleic acid fragment group c includes nucleic acid fragment mi and nucleic acid fragment mi +1 , wherein the 3'-end sequence of nucleic acid fragment mi and the 3'-end sequence of RNA fragment ni are complementary sequences, and nucleic acid fragment m
  • nucleic acid fragment m i+1 are complementary sequences; the 3'-end sequence of nucleic acid fragment m i+1 and the 3'-end sequence of RNA fragment n i+1 are complementary sequences.
  • the 5'-end sequence of the fragment mi+ 1 is a complementary sequence to the 5'-end sequence of the RNA fragment n i+2 or an unpaired sequence.
  • the nucleic acid fragment mi and the nucleic acid fragment mi+1 are independently DNA fragments or RNA fragments.
  • nucleic acid fragment group c includes nucleic acid fragment mi , nucleic acid fragment mi+1 , nucleic acid fragment mi+2 .
  • the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the RNA fragment n i are complementary sequences
  • the 5'-end sequence of the nucleic acid fragment mi and the 5'-end sequence of the RNA fragment n i+1 are complementary sequences
  • the 3'-end sequence of the fragment m i +1 is complementary to the 3'-end sequence of the RNA fragment n i+1
  • the 5'-end sequence of the nucleic acid fragment m i+1 is the 5'-end sequence of the RNA fragment n i+2 .
  • nucleic acid fragment mi+2 is a complementary sequence
  • the 3'-end sequence of the nucleic acid fragment mi+2 and the 3'-end sequence of the RNA fragment n i+2 are complementary sequences
  • the 5'-end sequence of the nucleic acid fragment mi+2 is the same as that of the RNA fragment n i+3
  • the 5'-end sequence is either a complementary sequence or an unpaired sequence.
  • the nucleic acid fragment m i , the nucleic acid fragment mi+1 , and the nucleic acid fragment mi+2 are independently DNA fragments or RNA fragments.
  • nucleic acid fragment group c includes nucleic acid fragment mi , nucleic acid fragment mi+1 , nucleic acid fragment mi+3 .
  • the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the RNA fragment n i are complementary sequences
  • the 5'-end sequence of the nucleic acid fragment mi and the 5'-end sequence of the RNA fragment n i+1 are complementary sequences
  • the 3'-end sequence of the fragment m i +1 is complementary to the 3'-end sequence of the RNA fragment n i+1
  • the 5'-end sequence of the nucleic acid fragment m i+1 is the 5'-end sequence of the RNA fragment n i+2 .
  • the 5'-end sequence of the nucleic acid fragment mi+3 is the complementary sequence; the 3'-end sequence of the nucleic acid fragment mi+3 is the complementary sequence with the 3'-end sequence of the RNA fragment n i+3 , and the 5'-end sequence of the nucleic acid fragment mi+3 is the same as the RNA fragment.
  • the 5'-end sequence of fragment n i+4 is a complementary sequence or an unpaired sequence.
  • nucleic acid fragment mi the nucleic acid fragment mi+1 , the nucleic acid fragment mi+2 , and the nucleic acid fragment mi+3 are independently DNA fragments or RNA fragments.
  • nucleic acid fragment group c may also include other numbers of nucleic acid fragments, which are not exhaustive in the present disclosure.
  • the RNA fragment group of the first strand is RNA fragment group a and RNA fragment group b
  • the nucleic acid fragment group of the second strand is nucleic acid fragment group c and nucleic acid fragment group d, that is, the RNA of the first strand
  • the sequence (that is, the sequence of the target long RNA) is divided into the sequence of RNA fragment n i , the sequence of RNA fragment n i+1 , the sequence of RNA fragment x ii and the sequence of RNA fragment x ii+1 , the sequence of RNA fragment x ii and the partial bases of RNA fragment x ii+1 are complementary to form a hairpin structure with 5' extension arm and 3' extension arm;
  • the nucleic acid sequence of the second strand is divided into the sequence of nucleic acid fragment m i , the sequence of nucleic acid fragment y ii
  • the sequence and the sequence of the nucleic acid fragment y ii+1 through the complementary pairing of the 5
  • the hairpin structure formed by the partial base complementation of RNA fragment x ii and RNA fragment x ii+1 also includes a stem region that forms a double-stranded structure and a stem-loop region that does not form a double-stranded structure.
  • the 5' end and the 3' end of the stem region are connected to the 5' extension arm and the 3' extension arm, respectively.
  • Whether a nucleic acid fragment group b including RNA fragment x ii and RNA fragment x ii+1 needs to be set in the first strand can be selected according to the spatial structure of the target long-chain RNA. For example, before the sequence division of nucleic acid fragments, the spatial structure of the target long-chain RNA is first predicted.
  • the sequence corresponding to the hairpin structure in the target long-chain RNA is divided.
  • the sequence corresponding to the linear structure in the target long-chain RNA is divided into RNA fragment ni and RNA fragment ni+1 .
  • sequence division is performed on the second strand complementary to the first strand, the nucleic acid fragment m i is divided corresponding to the RNA fragment n i and RNA fragment n i+1 of the first strand, and the nucleic acid fragment is divided corresponding to the hairpin structure of the first strand.
  • y ii nucleic acid fragment y ii+1 or a combination of nucleic acid fragment y ii and nucleic acid fragment y ii+1 .
  • RNA segment group b includes RNA segment xii and RNA segment xii+1 , and the first hairpin structure of the first strand is formed by RNA segment xii and RNA segment xii+1 .
  • RNA fragment group b includes RNA fragment xii, RNA fragment xii+1 , RNA fragment xii+2 , RNA fragment xii+3 , wherein RNA fragment xii , RNA fragment xii+1 The first hairpin structure of the first strand is formed, and the RNA fragment x ii+2 and the RNA fragment x ii+3 form the second hairpin structure of the first strand.
  • RNA fragment group b includes RNA fragment xii, RNA fragment xii+1 , RNA fragment xii+2 , RNA fragment xii+3 , RNA fragment xii+4 , RNA fragment xii+5 , wherein, RNA fragment x ii , RNA fragment x ii+1 form the first hairpin structure of the first strand, RNA fragment x ii+2 , RNA fragment x ii+3 form the second hairpin structure of the first strand, RNA Fragment xii+4 , RNA fragment xii+5 form the third hairpin structure of the first strand.
  • the RNA fragment group b may also include other numbers of nucleic acid fragments, and the specific number is set according to the number of hairpin structures in the target long-chain RNA, which is not exhaustive in the present disclosure.
  • nucleic acid fragment group d includes nucleic acid fragment y ii , the 5'-end sequence of nucleic acid fragment y ii is complementary to the sequence of the 5' extension arm of the first hairpin structure, and the 3'-end sequence of nucleic acid fragment y ii is complementary to the sequence of the 5' end of the nucleic acid fragment y ii.
  • the nucleic acid fragments in fragment group a form complementary sequences or unpaired sequences, and nucleic acid fragments y ii are DNA fragments or RNA fragments.
  • nucleic acid fragment group d includes nucleic acid fragment y ii+1 , the 3' end sequence of nucleic acid fragment y ii+1 is complementary to the 3' extension arm sequence of the first hairpin structure, and the nucleic acid fragment y ii+1 has a The 5'-end sequence and the nucleic acid fragment in the nucleic acid fragment group a form a complementary sequence or an unpaired sequence, and the nucleic acid fragment y ii+1 is a DNA fragment or an RNA fragment.
  • nucleic acid fragment group d includes nucleic acid fragment y ii and nucleic acid fragment y ii +1 , the 5' end sequence of nucleic acid fragment y ii is complementary to the 5' extension arm sequence of the first hairpin structure, and nucleic acid fragment y ii
  • the 3' end sequence of the nucleic acid fragment group a forms a complementary sequence or an unpaired sequence; the 3' end sequence of the nucleic acid fragment y ii+1 is complementary to the 3' extension arm sequence of the first hairpin structure, and the nucleic acid fragment
  • the 5'-end sequence of y ii+1 forms a complementary sequence with the nucleic acid fragment in the nucleic acid fragment group a or is an unpaired sequence.
  • the nucleic acid fragment y ii and the nucleic acid fragment y ii+1 are independently selected from DNA fragments or RNA fragments.
  • nucleic acid fragment group d includes nucleic acid fragment y ii , nucleic acid fragment y ii+1 and nucleic acid fragment y ii+2 , the 5' end sequence of nucleic acid fragment y ii and the 5' extension arm of the first hairpin structure
  • the sequences are complementary, the 3'-end sequence of the nucleic acid fragment y ii and the nucleic acid fragment in the nucleic acid fragment group a form a complementary sequence or an unpaired sequence; the 3'-end sequence of the nucleic acid fragment y ii+1 and the 3' of the first hairpin structure
  • the sequence of the extension arm is complementary, and the 5'-end sequence of the nucleic acid fragment y ii+1 forms a complementary sequence or an unpaired sequence with the nucleic acid fragment in the nucleic acid fragment group a; the 5'-end sequence of the nucleic acid fragment y ii+2 and the second hairpin
  • the 5' extension arm sequence of the structure is
  • nucleic acid fragment group d includes nucleic acid fragment y ii , nucleic acid fragment y ii+1 and nucleic acid fragment y ii+3 , the 5' end sequence of nucleic acid fragment y ii and the 5' extension arm of the first hairpin structure
  • the sequences are complementary, the 3'-end sequence of the nucleic acid fragment y ii and the nucleic acid fragment in the nucleic acid fragment group a form a complementary sequence or an unpaired sequence; the 3'-end sequence of the nucleic acid fragment y ii+1 and the 3' of the first hairpin structure
  • the sequence of the extension arm is complementary, and the 5'-end sequence of the nucleic acid fragment y ii+1 forms a complementary sequence or an unpaired sequence with the nucleic acid fragment in the nucleic acid fragment group a; the 3'-end sequence of the nucleic acid fragment y ii+3 and the second hairpin
  • the 3' extension arm sequence of the structure is
  • the nucleic acid fragment group d includes nucleic acid fragment y ii , nucleic acid fragment y ii+1 , nucleic acid fragment y ii+2 and nucleic acid fragment y ii+3 , and the 5'-end sequence of nucleic acid fragment y ii is the same as the first sequence.
  • the 5' extension arm sequence of the clip structure is complementary, and the 3' end sequence of the nucleic acid fragment y ii and the nucleic acid fragment in the nucleic acid fragment group a form a complementary sequence or an unpaired sequence; the 3' end sequence of the nucleic acid fragment y ii+1 is the same as the first
  • the 3' extension arm sequence of a hairpin structure is complementary, and the 5' end sequence of the nucleic acid fragment y ii+1 forms a complementary sequence or an unpaired sequence with the nucleic acid fragment in the nucleic acid fragment group a; the 5' end sequence of the nucleic acid fragment y ii+2
  • the end sequence is complementary to the 5' extension arm sequence of the second hairpin structure, and the 3' end sequence of the nucleic acid fragment y ii+2 forms a complementary sequence or an unpaired sequence with the nucleic acid fragment in the nucleic acid fragment group a; the nucleic acid fragment y ii+ The 3 ' end sequence of
  • nucleic acid fragment group b may also include other numbers of nucleic acid fragments, which are not exhaustive in the present disclosure.
  • nucleic acid fragment y ii , the nucleic acid fragment y ii+1 , the nucleic acid fragment y ii+2 , and the nucleic acid fragment y ii+3 are independently selected from DNA fragments or RNA fragments.
  • RNA fragment group b of the first strand and the nucleic acid fragment group d in the corresponding second strand is determined by the spatial structure of the target long-chain RNA; the actual number of RNA fragment group b and nucleic acid fragment group d is determined by Determined by the number of hairpin structures in the target long RNA.
  • the number of RNA fragment group a in the first strand and the number of nucleic acid fragment group c in the second strand are determined by the sequence of the target long-chain RNA to be synthesized. The increase or decrease of the number of fragments can realize the division of RNA chains of different lengths, and then realize the synthesis of long-chain RNAs of required length and required quantity.
  • the nucleic acid fragment group a is the RNA fragment group a
  • the nucleic acid fragment group c is the RNA fragment group c.
  • RNA fragment group a includes RNA fragment n i and RNA fragment n i +1
  • RNA fragment group c includes RNA fragment mi
  • the 5' end sequence of RNA fragment mi is complementary to the 5' end sequence of RNA fragment n i+1 sequence
  • the 3'-end sequence of RNA fragment mi and the 3'-end sequence of RNA fragment ni are complementary sequences.
  • RNA fragment group b includes RNA fragment x ii and RNA fragment x ii+1
  • RNA fragment group d includes RNA fragment y ii , RNA fragment y ii+1 or both RNA fragment y ii and RNA fragment y ii+1
  • the partial bases of RNA fragment x ii and RNA fragment x ii+1 are complementary to form a hairpin structure with a 5' extension arm and a 3' extension arm, the 5' extension arm sequence and the 5' end of RNA fragment y ii
  • the sequence is a complementary sequence
  • the 3' extension arm sequence and the 3' end sequence of the RNA fragment y ii+1 are complementary sequences.
  • the nucleic acid fragment group of the first strand is the RNA fragment group a
  • the nucleic acid fragment group of the second strand is the RNA fragment group c
  • the nucleic acid sequence of the first strand is divided into RNA fragments n i
  • the sequence and the sequence of the RNA fragment n i+1 , the nucleic acid sequence of the second strand is divided into the sequence of the RNA fragment m i formed by the 5' end sequence of the RNA fragment mi and the 5' end sequence of the RNA fragment n i+1
  • the complementary pairing of the 3'-end sequence of the RNA fragment mi and the 3'-end sequence of the RNA fragment ni realizes the sequence division of the double-stranded RNA containing the target long-chain RNA.
  • RNA fragment group a may also contain other RNA fragments.
  • RNA fragment group a includes RNA fragment n i , RNA fragment n i+1 , RNA fragment n i+2 .
  • RNA fragment set a includes RNA fragment n i , RNA fragment n i+1 , RNA fragment n i+2 , RNA fragment n i+3 .
  • RNA fragment group a includes RNA fragment n i , RNA fragment n i+1 , RNA fragment n i+2 , RNA fragment n i+3 , RNA fragment n i+4 .
  • the RNA fragment group a may also include other numbers of RNA fragments, which are not exhaustive in the present disclosure.
  • RNA fragment group c may also contain other RNA fragments.
  • RNA fragment group c includes RNA fragment m i and RNA fragment mi+1 , wherein the 3'-end sequence of RNA fragment mi and the 3'-end sequence of RNA fragment ni are complementary sequences, and RNA fragment m
  • the 5'-end sequence of i is complementary to the 5'-end sequence of RNA fragment n i+1 or is an unpaired sequence; the 3'-end sequence of RNA fragment m i+1 is the same as the 3'-end sequence of RNA fragment n i+1 .
  • the sequence is a complementary sequence, and the 5'-end sequence of the RNA fragment m i+1 is a complementary sequence or an unpaired sequence with the 5'-end sequence of the RNA fragment n i+2 .
  • RNA fragment group c includes RNA fragment mi , RNA fragment mi+1 , RNA fragment mi+2 .
  • the 3'-end sequence of RNA fragment mi and the 3'-end sequence of RNA fragment ni are complementary sequences, and the 5'-end sequence of RNA fragment mi is complementary to the 5'-end sequence of RNA fragment ni +1 or is an unpaired sequence; the 3'-end sequence of RNA fragment m i+1 and the 3'-end sequence of RNA fragment n i+1 are complementary sequences, and the 5'-end sequence of RNA fragment mi+1 is the same as that of RNA fragment n i+
  • the 5'-end sequence of 2 is a complementary sequence; the 3'-end sequence of RNA fragment m i+2 and the 3'-end sequence of RNA fragment n i+2 are complementary sequences, and the 5'-end sequence of RNA fragment mi+2 is the same as The 5'-end sequence of the RNA fragment n i+3 is either a
  • RNA fragment group c includes RNA fragment mi , RNA fragment mi+1 , RNA fragment mi+3 .
  • the 3'-end sequence of RNA fragment mi and the 3'-end sequence of RNA fragment ni are complementary sequences, and the 5'-end sequence of RNA fragment mi is complementary to the 5'-end sequence of RNA fragment ni +1 or is an unpaired sequence; the 3'-end sequence of RNA fragment m i+1 and the 3'-end sequence of RNA fragment n i+1 are complementary sequences, and the 5'-end sequence of RNA fragment mi+1 is the same as that of RNA fragment n i+
  • the 5'-end sequence of 2 is a complementary sequence; the 3'-end sequence of RNA fragment m i+2 and the 3'-end sequence of RNA fragment n i+2 are complementary sequences, and the 5'-end sequence of RNA fragment mi+2 is the same as The sequence at the 5' end of the RNA fragment n i+3 is a
  • the nucleic acid fragment groups of the first strand are RNA fragment group a and RNA fragment group b
  • the nucleic acid fragment groups of the second strand are RNA fragment group c and RNA fragment group d, that is, the first strand
  • the nucleic acid sequence is divided into the sequence of RNA fragment n i , the sequence of RNA fragment n i+1 , the sequence of RNA fragment x ii and the sequence of RNA fragment x ii+1 , the sequence of RNA fragment x ii and RNA fragment x ii+1 Partial base complementation forms a hairpin structure with a 5' extension arm and a 3' extension arm;
  • the nucleic acid sequence of the second strand is divided into the sequence of RNA fragment mi, the sequence of RNA fragment y ii , and the sequence of RNA fragment y ii +1 .
  • Sequence through the complementary pairing of the 5' end sequence of the RNA fragment mi and the 5' end sequence of the RNA fragment n i +1 , the complementary pairing of the 3' end sequence of the RNA fragment mi and the 3' end sequence of the RNA fragment n i , the complementary pairing of the 5' end sequence of the RNA fragment y ii and the 5' extension arm sequence, the complementary pairing of the 3' end sequence of the RNA fragment y ii+1 and the 3' extension arm sequence, to achieve the target length of the hairpin structure. Sequence division of double-stranded RNA from stranded RNA.
  • the hairpin structure formed by the partial base complementation of RNA fragment x ii and RNA fragment x ii+1 also includes a stem region that forms a double-stranded structure and a stem-loop region that does not form a double-stranded structure.
  • the 5' end and the 3' end of the stem region are connected to the 5' extension arm and the 3' extension arm, respectively.
  • Whether the RNA fragment group b including the RNA fragment x ii and the RNA fragment x ii+1 needs to be set in the first strand can be selected according to the spatial structure of the target long-chain RNA. For example, before the sequence division of RNA fragments, the spatial structure of the target long-chain RNA is first predicted.
  • the sequence of the target long-chain RNA is designed as the first strand. , the sequence corresponding to the hairpin structure in the target long-chain RNA is divided into RNA fragment x ii and RNA fragment x ii+1 , and the sequence corresponding to the linear structure in the target long-chain RNA is divided into RNA fragment n i and RNA fragment n i+ 1 .
  • the second strand complementary to the first strand is divided into sequences, the RNA fragments n i and n i+1 of the corresponding first strand are divided into m i , and the hairpin structure of the first strand is divided into RNA fragments y ii , RNA Fragment y ii+1 or a combination of RNA fragment y ii and RNA fragment y ii+1 .
  • RNA fragment group b includes RNA fragment xii and RNA fragment xii+1 , and the first hairpin structure of the first strand is formed by RNA fragment xii and RNA fragment xii+1 .
  • RNA fragment group b includes RNA fragment xii, RNA fragment xii+1 , RNA fragment xii+2 , RNA fragment xii+3 , wherein RNA fragment xii , RNA fragment xii+1 The first hairpin structure of the first strand is formed, and the RNA fragment x ii+2 and the RNA fragment x ii+3 form the second hairpin structure of the first strand.
  • RNA fragment group b includes RNA fragment xii, RNA fragment xii+1 , RNA fragment xii+2 , RNA fragment xii+3 , RNA fragment xii+4 , RNA fragment xii+5 , wherein, RNA fragment x ii , RNA fragment x ii+1 form the first hairpin structure of the first strand, RNA fragment x ii+2 , RNA fragment x ii+3 form the second hairpin structure of the first strand, RNA Fragment xii+4 , RNA fragment xii+5 form the third hairpin structure of the first strand.
  • the RNA fragment group b may also include other numbers of RNA fragments, and the specific number is set according to the number of hairpin structures in the target long-chain RNA, which is not exhaustive in this disclosure.
  • RNA fragment group d includes RNA fragment y ii , the 5' end sequence of RNA fragment y ii is complementary to the 5' extension arm sequence of the first hairpin structure, and the 3' end sequence of RNA fragment y ii Complementary or unpaired sequences to RNA fragments in RNA fragment group a.
  • RNA fragment group d includes RNA fragment y ii+1 , the 3' end sequence of RNA fragment y ii+1 is complementary to the sequence of the 3' extension arm of the first hairpin structure, and the sequence of RNA fragment y ii+1 is The 5'-end sequence forms a complementary sequence with the RNA fragment in RNA fragment group a or is an unpaired sequence.
  • RNA fragment group d includes RNA fragment y ii and RNA fragment y ii+1 , the 5'-end sequence of RNA fragment y ii is complementary to the sequence of the 5' extension arm of the first hairpin structure, and the RNA fragment
  • the 3'-end sequence of y ii and the RNA fragment in the RNA fragment group a form a complementary sequence or an unpaired sequence;
  • the 3'-end sequence of the RNA fragment y ii+1 is complementary to the 3' extension arm sequence of the first hairpin structure,
  • the 5'-end sequence of the RNA fragment y ii+1 forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a.
  • the RNA segment group d includes RNA segment y ii , RNA segment y ii+1 and RNA segment y ii+2 , the 5' end sequence of RNA segment y ii and the 5' end sequence of the first hairpin structure
  • the sequence of the extension arm is complementary, and the 3'-end sequence of the RNA fragment y ii forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a; the 3'-end sequence of the RNA fragment y ii+1 is the same as that of the first hairpin structure.
  • the 3' extension arm sequence is complementary, and the 5' end sequence of the RNA fragment y ii+1 forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a; the 5' end sequence of the RNA fragment y ii+2 is the same as the second sequence.
  • the 5' extension arm sequence of the hairpin structure is complementary, and the 3' end sequence of the RNA fragment y ii+2 forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a.
  • the RNA segment group d includes RNA segment y ii , RNA segment y ii+1 and RNA segment y ii+3 , the 5'-end sequence of RNA segment y ii and the 5' of the first hairpin structure
  • the sequence of the extension arm is complementary, and the 3'-end sequence of the RNA fragment y ii forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a; the 3'-end sequence of the RNA fragment y ii+1 is the same as that of the first hairpin structure.
  • the 3' extension arm sequence is complementary, and the 5' end sequence of the RNA fragment y ii+1 forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a; the 3' end sequence of the RNA fragment y ii+3 is the same as the second sequence.
  • the 3' extension arm sequence of the hairpin structure is complementary, and the 5' end sequence of the RNA fragment y ii+3 forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a.
  • group d includes y ii , y ii+1 , y ii+2 and y ii+3 , the sequence of the 5' end of the RNA fragment y ii and the sequence of the 5' extension arm of the first hairpin structure Complementary, the 3' end sequence of the RNA fragment y ii and the RNA fragment in the RNA fragment group a form a complementary sequence or an unpaired sequence; the 3' end sequence of the RNA fragment y ii+1 and the 3' extension of the first hairpin structure
  • the arm sequences are complementary, and the 5'-end sequence of the RNA fragment y ii+1 forms a complementary sequence or an unpaired sequence with the RNA fragment in the RNA fragment group a; the 5'-end sequence of the RNA fragment y ii+2 and the second hairpin structure
  • the 5' extension arm sequence is complementary, and the 3' end sequence of the RNA fragment y ii+2 forms a complementary sequence or an unpaired sequence with
  • the 5'-end sequence and the 3'-end sequence refer to dividing the nucleotide fragment in the 5' to 3' direction, so that the nucleotide fragment is divided into two regions.
  • a sequence in a region near the 5' end is referred to as a 5' end sequence
  • a sequence in another region near the 3' end is referred to as a 3' end sequence.
  • the 5' terminus is a nucleotide in the 5' to 3' direction at the 5' endmost position in the nucleotide chain, which typically has a 5' terminus phosphate group.
  • the 3' terminus is a nucleotide in the 5' to 3' direction at the 3' endmost position in the nucleotide chain, which typically has a 3' terminus hydroxyl group.
  • nucleotide sequences of the first strand and the second strand are divided, there will be a junction between the two connected nucleic acid fragments.
  • a junction between RNA fragment n i and RNA fragment n i+1 in the first strand a junction between RNA fragment x ii and RNA fragment x ii+1 in the first strand, and a nucleic acid fragment in the second strand
  • a junction exists between nucleic acid fragment y ii and nucleic acid fragment y ii +1 .
  • the adjacent nucleic acids in the nucleic acid fragment group of the first strand are staggered from each other.
  • the junction between RNA fragment xii and RNA fragment xii+1 is located in the stem region of the hairpin structure.
  • RNA sequence with secondary structure into RNA fragments x ii and RNA fragments x ii+1 , and using the target RNA sequence to form a hairpin structure by itself, it is possible to maximize the retention of the target RNA sequence formed after the connection is completed.
  • the high-level structure of single-stranded long RNAs especially RNA sequences with special physiological functions, so as to ensure that their physiological functions are not affected.
  • the melting temperature (T m ) of the nucleic acid fragments in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand should be as close as possible, And avoid the existence of complex high-level structures in the chain, so as to reduce the difficulty of annealing nucleic acid fragments to form double-stranded assembly precursors.
  • the length of the 5'-end sequence of any nucleic acid fragment in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 4 nt or more, preferably 4-50 nt, more preferably 6-30 nt, Most preferably 10-20nt.
  • the 5' end sequence of any nucleic acid fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the nucleic acid fragment group of the first strand is an RNA fragment group
  • the length of the 5'-end sequence of any RNA fragment in the RNA fragment group is 4 nt or more, preferably 4-50 nt, more preferably 6- 30nt, most preferably 10-20nt.
  • the 5' end sequence of any RNA fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the set of nucleic acid fragments of the second strand is a set of RNA fragments, a set of DNA fragments, or a combination of RNA fragments and DNA fragments, wherein the 5'-end sequence of any RNA fragment or any DNA fragment
  • the length is 4nt or more, preferably 4-50nt, more preferably 6-30nt, most preferably 10-20nt.
  • the 5' end sequence of any RNA fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the length of the 3' end sequence of any nucleic acid fragment in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 4 nt or more, preferably 4-50 nt, more preferably 6-30 nt, Most preferably 10-20nt.
  • the 3' end sequence of any nucleic acid fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the nucleic acid fragment group of the first strand is an RNA fragment group
  • the length of the 3' end sequence of any RNA fragment in the RNA fragment group is 4 nt or more, preferably 4-50 nt, more preferably 6- 30nt, most preferably 10-20nt.
  • the 3' end sequence of any RNA fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the set of nucleic acid fragments of the second strand is a set of RNA fragments, a set of DNA fragments, or a combination of RNA fragments and DNA fragments, wherein the sequence of the 3' end of any RNA fragment or any DNA fragment
  • the length is 4nt or more, preferably 4-50nt, more preferably 6-30nt, most preferably 10-20nt.
  • the 3' end sequence of any RNA fragment is 4nt, 6nt, 8nt, 10nt, 12nt, 14nt, 16nt, 18nt, etc. in length.
  • the length of the continuous single-stranded RNA is 60-1000 nt, preferably 80-600 nt, more preferably 100-400 nt, most preferably 120-360 nt.
  • the length of any single stranded nucleic acid strand is 60nt, 70nt, 80nt, 90nt, 100nt, 120nt, 140nt, 160nt, 180nt, 200nt, 220nt, 240nt, 250nt, 260nt, 267nt, 270nt, 300nt, 320nt, 340nt, 360nt , 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, etc.
  • the first strand is a single-stranded RNA assembled from RNA fragments, and after connecting the connecting ports in the first strand, a continuous single-stranded RNA is obtained, and the length of the continuous single-stranded RNA is 60-1000nt, preferably 80-600nt, more preferably 100-400nt, most preferably 120-360nt.
  • the lengths of single-stranded RNA are 60nt, 70nt, 80nt, 90nt, 100nt, 120nt, 140nt, 160nt, 180nt, 200nt, 220nt, 240nt, 250nt, 260nt, 267nt, 270nt, 300nt, 320nt, 340nt, 360nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, etc.
  • the second strand is single-stranded RNA, single-stranded DNA, or a mixed single-stranded DNA and RNA, and the second strand is present in a double-stranded assembly and is a fragmented single-stranded nucleic acid strand.
  • the length of the second strand in the double-stranded assembly is 60-1000 nt, preferably 80-600 nt, more preferably 100-400 nt, most preferably 120-360 nt.
  • the lengths of single-stranded RNA are 60nt, 70nt, 80nt, 90nt, 100nt, 120nt, 140nt, 160nt, 180nt, 200nt, 220nt, 240nt, 250nt, 260nt, 267nt, 270nt, 300nt, 320nt, 340nt, 360nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, etc.
  • the present disclosure describes a method for preparing single-stranded long-stranded RNA, comprising the following steps:
  • Synthesis step Synthesize the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand, the nucleic acid fragment group of the first strand is composed of RNA fragments, and the nucleic acid fragment group of the second strand is composed of RNA fragments and DNA fragments. at least one of the constituents;
  • the nucleic acid fragment group of the first strand includes an RNA fragment group a and an optional RNA fragment group b
  • the nucleic acid fragment group of the second strand includes a nucleic acid fragment group c and an optional nucleic acid fragment group d
  • the RNA fragment Group a includes RNA fragments n i and RNA fragments n i+1
  • said nucleic acid fragment group b includes RNA fragments x ii and RNA fragments x ii+1
  • said nucleic acid fragment group c includes nucleic acid fragments mi
  • said nucleic acid fragments Group d includes at least one of nucleic acid fragments y ii and nucleic acid fragments y ii+1 , where i and ii are independently selected from integers of 1 or more;
  • the 5'-end sequence of the nucleic acid fragment mi and the 5'-end sequence of the RNA fragment n i +1 are complementary sequences
  • the 3'-end sequence of the nucleic acid fragment mi and the 3'-end sequence of the RNA fragment n i are complementary sequences
  • Annealing step mixing the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand in the same reaction system and annealing to form a double-stranded assembly precursor; There is a junction between each nucleic acid fragment, and a junction exists between two adjacent nucleic acid fragments in the second strand; the junction between the adjacent nucleic acid fragments in the nucleic acid fragment group of the first strand is the same as the The connection ports between adjacent nucleic acid fragments in the nucleic acid fragment group of the second strand are staggered from each other;
  • Connecting step connecting the connecting port of the first strand to obtain a double-stranded assembly formed by the complementation of the continuous single-stranded RNA and the fragmented single-stranded nucleic acid strand.
  • Denaturation step denaturation of the double-stranded assembly to obtain a continuous single-stranded RNA.
  • the synthesis method of nucleic acid fragments can adopt the RNA and DNA synthesis methods commonly used in the art, for example, solid phase synthesis.
  • the solid-phase synthesis method can prepare short-chain nucleic acid fragments on a large scale, and the sequence accuracy of the nucleic acid fragments can be ensured.
  • the solid-phase synthesis method that can realize the introduction of specific position modification is usually only used for the synthesis of short-chain RNAs below 60 nt, and the longest is no more than 120 nt, and the solid-phase synthesis method is used to prepare the length of the RNA. It is difficult to introduce site-directed modification for short-strand RNAs in the range of 60-120 nt; however, in vitro transcription and RCT methods suitable for long-strand RNA synthesis are difficult to introduce site-directed modification.
  • the length of any nucleic acid fragment in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 8-120nt, preferably 10-80nt, more preferably 15-40nt, most preferably 20-nt 30nt.
  • nucleic acid fragments are 22nt, 24nt, 26nt, 28nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, etc. in length.
  • the length of nucleic acid fragments determines the difficulty and cost of their synthesis. Controlling the length of nucleic acid fragments at 20-30 nt can effectively reduce the difficulty of synthesizing nucleic acid fragments and control the cost of synthesis.
  • the nucleic acid fragment group of the first strand is an RNA fragment group, and the length of any RNA fragment in the RNA fragment group is 8-120nt, preferably 10-80nt, more preferably 15-40nt, most preferably 20-30nt.
  • RNA fragments are 22nt, 24nt, 26nt, 28nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, etc. in length.
  • the group of nucleic acid fragments of the second strand is a group of RNA fragments, a group of DNA fragments, or a combination of RNA fragments and DNA fragments, wherein the length of any RNA fragment or any DNA fragment is 8-120 nt , preferably 10-80nt, more preferably 15-40nt, most preferably 20-30nt.
  • RNA fragments are 22nt, 24nt, 26nt, 28nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, etc. in length.
  • modified bases are included at one or more positions in any of the nucleic acid fragments in the set of nucleic acid fragments of the first strand and the set of nucleic acid fragments of the second strand.
  • modified bases are included at 1, 2, 3, 4, etc. positions of the nucleic acid fragment.
  • the method of base modification can adopt methods commonly used in the art, for example, introducing modified bases in the process of chemically synthesizing short-chain nucleic acid fragments. Introducing modified bases in the process of synthesizing nucleic acid fragments can realize base modification at any site. After the nucleic acid fragments are assembled into long-chain RNAs, long-chain RNAs that can precisely modify bases at any site can be obtained. .
  • the modification of the base at any position in the nucleic acid fragment can be selected from m 6 A, ⁇ , m 1 A, m 5 A, ms 2 i 6 A, i 6 A, m 3 C, m 5 C, ac 4 C, m 7 G, m2, 2G, m 2 G, m 1 G, Q, m 5 U, mcm 5 U, ncm 5 U, ncm 5 Um, D, mcm 5 s 2 U, Inosine ( I), hm 5 C, s 4 U, s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or derivatives thereof [19,20] .
  • the set of nucleic acid fragments of the first strand is a set of RNA fragments, and any RNA fragment in the set of RNA fragments comprises modified ribose sugars at one or more positions.
  • modified bases are included at 1, 2, 3, 4, etc. positions of the RNA fragment.
  • modified ribose sugars are included at one or more positions in any of the set of nucleic acid fragments of the first strand and the set of nucleic acid fragments of the second strand.
  • modified ribose sugars are included at 1, 2, 3, 4, etc. positions of the nucleic acid fragment.
  • the method of ribose modification can adopt the method commonly used in the art, for example, introducing modified ribose in the process of chemical synthesis of short-chain nucleic acid fragments. The introduction of modified ribose in the process of synthesizing nucleic acid fragments can realize the modification of ribose at any site. After the nucleic acid fragment is assembled into long-chain RNA, long-chain RNA that can precisely modify ribose at any site can be obtained.
  • the modification mode of ribose at any position in the nucleic acid fragment can be selected from LNA, 2'-OMe, 3'-OMeU, vmoe, 2'-F or 2'-OBn (2'-O-benzyl group) or its derivatives [21] .
  • the set of nucleic acid fragments of the first strand is a set of RNA fragments, and any RNA fragment in the set of RNA fragments comprises modified ribose sugars at one or more positions.
  • modified ribose sugars are included at 1, 2, 3, 4, etc. positions of the RNA fragment.
  • the set of nucleic acid fragments of the first strand and the set of nucleic acid fragments of the second strand comprise modified phosphodiester bonds at one or more positions, the phosphodiester bonds being formed in the short Between two adjacent nucleotides of a stranded nucleic acid fragment.
  • modified phosphodiester linkages are included at 1, 2, 3, 4, etc. positions of the nucleic acid fragment.
  • the method of phosphodiester bond modification can adopt methods commonly used in the art, for example, introducing modified phosphodiester bonds in the process of chemically synthesizing short-chain nucleic acid fragments. Introducing modified phosphodiester bonds in the process of synthesizing nucleic acid fragments can realize the modification of phosphodiester bonds at any site. Precisely modified long RNAs.
  • the modification of the phosphodiester bond at any position in the nucleic acid fragment can be selected from Phosphorothioate (PS), nucleotide triphosphate (NTP ⁇ S) or derivatives thereof [22,23] .
  • PS Phosphorothioate
  • NTP ⁇ S nucleotide triphosphate
  • modifications to the base, ribose, and phosphodiester linkages should avoid the base, ribose, and phosphodiester linkages immediately adjacent to the junction to avoid first-strand or second-strand junctions Modifications at these sites may affect the junction junctions in the assembly precursors of subsequent double-stranded RNAs.
  • the nucleic acid fragment group of the first strand is an RNA fragment group, and one or more positions of any RNA fragment in the RNA fragment group comprise modified phosphodiester bonds, and the phosphodiester bonds form between two adjacent ribonucleotides of short nucleic acid fragments.
  • modified phosphodiester linkages are included at 1, 2, 3, 4, etc. positions of the nucleic acid fragment.
  • the modified nucleic acid fragment By modifying at least one of the base, ribose and phosphodiester bonds at any one or more sites in the nucleic acid fragment, and applying the modified nucleic acid fragment to the synthesis of long-chain RNA in the present disclosure, the The precise modification of any site in the long-chain RNA effectively solves the problem that it is difficult to synthesize the long-chain RNA with precise modification of the specific site in the current field.
  • the modified long-chain RNA not only improves the structural stability, but also further improves the biological properties such as the immunogenicity of the long-chain RNA, so that the synthesized long-chain RNA can be widely used in the field of biomedicine.
  • the nucleic acid fragment group of the first strand is an RNA fragment group
  • any RNA fragment in the RNA fragment group comprises a phosphate group at the 5' end and a hydroxyl group at the 3' end.
  • the 5' end of RNA fragment ni contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of RNA fragment ni+1 contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of RNA fragment ni+2 contains a phosphate group.
  • the 'end contains a phosphate group, and the 3' end contains a hydroxyl group; the 5' end of RNA fragment n i+3 contains a phosphate group, and the 3' end contains a hydroxyl group; the 5' end of RNA fragment x ii contains a phosphate group, and the 3' end contains a phosphate group Contains hydroxyl groups; when the nucleic acid fragments of the first and second strands are assembled to form a double-stranded assembly precursor with a hairpin structure, the 5' phosphate groups and 3' hydroxyl groups on both sides of the connecting port are connected to phosphodiester The bond can realize the connection of the connecting port in the first strand, so as to obtain a double-stranded assembly formed by the complementarity of the continuous single-stranded RNA (first strand) and the fragmented single-stranded nucleic acid strand (second strand).
  • the nucleic acid fragment group of the first strand is an RNA fragment group
  • any RNA fragment in the RNA fragment group comprises a phosphate group at the 5' end and a hydroxyl group at the 3' end.
  • the 5' end of RNA fragment ni contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of RNA fragment ni+1 contains a phosphate group and the 3' end contains a hydroxyl group
  • the 5' end of RNA fragment ni+2 contains a phosphate group.
  • the 'end contains a phosphate group and the 3'end contains a hydroxyl group; the 5'end of the RNA fragment n i+3 contains a phosphate group and the 3'end contains a hydroxyl group.
  • connection of the connecting port in the first strand can be realized, thereby obtaining a double-stranded assembly formed by complementation of the continuous single-stranded RNA (first strand) and the fragmented single-stranded nucleic acid strand (second strand).
  • the method for introducing the phosphate group at the 5' end of the RNA fragment can adopt a modification method commonly used in the art, for example, a phosphate group can be directly introduced at the 5' end of the RNA fragment during the synthesis of the RNA fragment; Alternatively, the RNA fragments without phosphate groups are subjected to kinase treatment to modify the 5' ends of the RNA fragments with phosphate groups.
  • the 5' phosphate group and the 3' hydroxyl group are added in the RNA fragments of the target single-stranded RNA in the first and second strands, and then only the target single-stranded RNA is connected in the connecting step.
  • the fragmented nucleic acid chain may be a nucleic acid chain composed of RNA fragments, a nucleic acid chain composed of DNA fragments, or a nucleic acid chain composed of both RNA fragments and DNA fragments.
  • nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand in the same reaction system and annealing to obtain a double-stranded assembly precursor formed by at least partial complementarity of the first strand and the second strand; wherein, There is a junction between two adjacent nucleic acid fragments in the first strand, and a junction exists between two adjacent nucleic acid fragments in the second chain; the phase in the nucleic acid fragment group of the first chain is The connection ports between adjacent nucleic acid fragments and the connection ports between adjacent nucleic acid fragments in the nucleic acid fragment group of the second strand are staggered from each other.
  • DNA molecules contain 4 kinds of deoxyribonucleotides, which are adenine deoxyribonucleotide (A), guanine deoxyribonucleotide (G), cytosine deoxyribonucleotide ( C) and thymidine (T). Similar to DNA, which contains 4 kinds of deoxyribonucleotides, RNA molecules contain 4 different kinds of ribonucleotides, which are adenine ribonucleotides (A) and guanine ribonucleotides, depending on the type of base. (G), cytosine ribonucleotides (C) and uracil ribonucleotides (U).
  • A adenine deoxyribonucleotide
  • G guanine deoxyribonucleotide
  • C cytosine deoxyribonucleotide
  • U uracil ribonucleotides
  • the bases can be connected to each other through hydrogen bonds, wherein A and T, A and U, and C and G can respectively form hydrogen bonds.
  • the precise complementary pairing ability between base pairs enables two reverse nucleic acid single strands whose sequences are complementary to each other to form an accurate double-stranded structure by hydrogen bonding.
  • the nucleic acid fragment of the first strand and the nucleic acid fragment of the second strand in the reaction system can be reassembled into the original target long double-stranded structure under the guidance of the principle of complementary base pairing after annealing .
  • the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand are dissolved in the same solvent, and the two are thoroughly mixed to obtain a reaction system for preparing a double-stranded assembly precursor.
  • the present disclosure does not specifically limit the specific solvent, which can be a polar solvent commonly used in the art, such as water and the like.
  • the molar ratio of any two nucleic acid fragments in the nucleic acid fragment group of the first strand and the nucleic acid fragment group of the second strand is 1:(0.1-10), preferably 1:( 0.5-2), most preferably 1:1.
  • the molar ratio of any two nucleic acid fragments is 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:4, 1:6, 1:8, etc.
  • the assembly efficiency of short-chain nucleic acid fragments can be improved.
  • the first-strand RNA fragment group and the second-strand RNA fragment group are dissolved in the same solvent, and the two are thoroughly mixed to obtain a reaction for preparing a double-stranded RNA assembly precursor system.
  • the present disclosure does not specifically limit the specific solvent, which can be a polar solvent commonly used in the art, such as water and the like.
  • the specific solvent can be a polar solvent commonly used in the art, such as water and the like.
  • the molar ratio of any two RNA fragments in the RNA fragment group of the first strand and the RNA fragment group of the second strand is 1:(0.1-10), preferably 1:( 0.5-2), most preferably 1:1.
  • the molar ratio of any two RNA fragments is 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:4, 1:6, 1:8, etc.
  • the assembly efficiency of short RNA fragments can be improved.
  • the pH of the reaction system is set to 3-11, preferably pH 4-10, more preferably pH 5-9, most preferably pH 6-8.
  • the pH of the reaction system is 6, 7, 8, 9, and the like.
  • the temperature is lowered to form a double-stranded assembly precursor
  • the incubation temperature is any temperature of 0-100°C, preferably any temperature of 10-85°C, more preferably any temperature in the interval of 20-65°C, and the incubation time is any desired time.
  • the speed of cooling can be any speed, and the temperature can be lowered to any temperature at which the nucleic acid fragments in the reaction system are hybridized to form the precursor of the double-stranded assembly.
  • the 5' phosphate groups and the 3' hydroxyl groups on both sides of the connection port are connected as phosphodiester bonds.
  • the ligation method may be enzymatic ligation in which T4 RNA ligase is ligated. or chemical linking. After the ligation port is connected, a complete double-stranded assembly formed by the complementarity of the first strand and the second strand is obtained.
  • the first strand in the double-stranded assembly is a continuous single-stranded RNA
  • the second strand is an RNA fragment, DNA fragment or RNA. Fragmented single-stranded nucleic acid chain composed of fragments and DNA fragments, realizes the preparation of long-chain RNA.
  • the preparation method of the present disclosure further includes a denaturation step.
  • the denaturation step the double-stranded assembly is denatured to obtain a continuous single-stranded RNA, that is, the target long-chain RNA.
  • the method of denaturation treatment can be a method commonly used in the art for melting double-stranded RNA to form single-stranded RNA.
  • single-stranded RNA is obtained by treating at 70°C for 5 min.
  • the preparation method of the present disclosure also includes a purification step.
  • the purification step is to purify the continuous single-stranded RNA from the reaction system.
  • the present disclosure does not specifically limit the purification method, and it can be various methods for efficiently recovering RNA from the reaction system.
  • the long-chain RNA without other substances obtained after the purification step can be further applied in different fields such as clinical, drug development, biological research and the like.
  • the preparation method in the present disclosure has all the advantages of conventional RNA chemical synthesis methods (including no need for template strands, precise site modification, etc.)
  • the complementary single-stranded nucleic acid strand of the single-stranded RNA can be divided into several shorter RNA fragments, DNA fragments, or a combination of RNA fragments and DNA fragments.
  • the difficulty of chemical synthesis is greatly reduced, and the high accuracy, high yield and site-specific modification ability of short-chain nucleic acid fragments prepared by chemical synthesis are retained.
  • nucleic acid fragments that can be easily prepared by solid-phase synthesis are reassembled into the double-stranded assembly precursor of the target structure in a specific order through the self-assembly ability of nucleic acid, and the connecting ports in the assembly are connected by techniques such as enzymatic linkage or chemical linkage. Religation through phosphodiester bonds results in a double-stranded assembly formed by the complementarity of the continuous single-stranded RNA and the fragmented single-stranded nucleic acid strand. For double-stranded assemblies, only simple denaturation is required to obtain single-stranded target long-chain RNAs.
  • the obtained target long-chain RNA also has the ability to be accurately modified at almost any position. Modified properties.
  • a second aspect of the present disclosure provides a long-chain RNA prepared by the method of the first aspect.
  • the long-chain RNA is a single-stranded long-chain RNA.
  • the long-chain RNA of the present disclosure can realize accurate modification at any site, and the long-chain RNA itself has no sequence dependence on the modification, so as to expand the long-chain RNA (especially the long-chain RNA with precise modification) in the field of biomedicine application provides the basis.
  • experimental techniques and experimental methods used in the present embodiment are conventional technical methods, such as experimental methods that do not specify specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer. Materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • RNA sequences used in the examples were all purchased from Ruipoxingke Company without additional treatment before use. All experimental water used ultrapure water produced by 18.2M ⁇ cm Millipore Company. T4 RNA ligase 2 and 10X ligase buffer were purchased from NEB Company. All other chemical reagents were of analytical grade.
  • the first and second strands are respectively 80 nt and 100 nt long RNA (referred to as RNA80/100) as the target long chain, and the long chain is cut into 9 short 20 nt long RNAs.
  • R-n1, R-n2, R-n3 and R-n4 are the RNA fragments for the synthesis of the first strand
  • R-m1, R-m2, R-m3, R-m4 and R-m5 are for the synthesis of the second strand RNA fragments.
  • R-n1, R-n2, R-n3 and R-n4, which are split from the first strand (80nt), are modified with a phosphate group at the 5' end, and R-m1 and R are split from the second strand.
  • -m2, R-m3, R-m4 and R-m5 are not modified.
  • the 5' terminal phosphate group was introduced during the synthesis of R-n1, R-n2, R-n3 and R-n4.
  • Example 3 According to the method in Example 1, an aqueous solution of RNA80/100 assembly was obtained, and its concentration was 10 ⁇ M. Different amounts of T4 RNA ligase (10, 20, 40 or 80U) and 2 ⁇ L of 10X T4 RNA ligase buffer were added to 10 ⁇ L of the assembly aqueous solution, and the total volume was 20 ⁇ L with ultrapure water. The system was reacted at 37°C for 10 min to connect the 3 junction sites in the first strand with T4 RNA ligase, so that the 4 short chains in the first strand formed a complete 80nt chain.
  • T4 RNA ligase 10, 20, 40 or 80U
  • 2 ⁇ L of 10X T4 RNA ligase buffer were added to 10 ⁇ L of the assembly aqueous solution, and the total volume was 20 ⁇ L with ultrapure water.
  • the system was reacted at 37°C for 10 min to connect the 3 junction sites in the first strand with T4 RNA liga
  • Example 3 In order to synthesize longer RNA sequences, the system was expanded in Example 3 to increase the number of short-chain RNAs. Specific steps are as follows:
  • RNA200/180 The first and second strands of long RNAs with lengths of 200 nt and 180 nt (referred to as RNA200/180) were used as the target long chain, and the long chain was cut into 19 short-chain RNAs with a length of 20 nt. Starting from the 5' end of strand 1, 10, 13 and 19 short strands were taken for assembly (referred to as RNA100/100, RNA140/120 and RNA200/180, respectively).
  • RNA fragments divided by the first strand include: R-n5, R-n6, R-n7, R-n8, R-n9, R-n10, R-n11, R-n12, R- n13, R-n14;
  • RNA fragments divided by the second strand include: R-m6, R-m7, R-m8, R-m9, R-m10, R-m11, R-m12, R- m13, R-m14.
  • Example 3 (1) Among the three groups of target RNA long chains of RNA100/100, RNA140/120 and RNA200/180 mentioned in Example 3, 5' of the 20 nt RNA fragments obtained by dividing the first strand (100 nt, 140 nt and 200 nt) The terminal phosphate group was modified, and the modification method was the same as that in Example 2, and the RNA fragments obtained by dividing the second strand (100nt, 120nt and 180nt) were not modified.
  • RNA100/80 assembly aqueous solution obtain RNA100/80 assembly aqueous solution, then connect 4, 6 and 9 connection port sites in chain 1 by T4 RNA ligase, and the connection method is the same as in embodiment 2, All RNA fragments in the first strand were made to form complete 100nt, 140nt and 200nt strands, respectively.
  • RNA fragment in step (2) is subjected to denaturation treatment, and the denaturation treatment method is the same as that of Example 2, to obtain a continuous first strand, and the first strand is observed by polyacrylamide gel electrophoresis, and the result is shown in Figure 7 Show.
  • RNA single strands of different lengths can be prepared in one pot, and the lengths of the shown RNA single strands are 100, 140 and 200 nt, respectively.
  • RNA duplexes up to 267/220 bp were split in Example 5. Details as follows:
  • RNA fragments of the first strand include R-n15, R-n16, R-n17, R-n18, R-x1, R-x2, R-n19, R-n20, R-n21, R-n22; split the second chain with a length of 220nt into 8 short chains with a length of 24-30nt etc.
  • the RNA fragments of the second strand include R-m15, R-m16, R-y1, R-y2, R-m17, R-m18, R-m19, R-m20 in Table 3.
  • the 5'-terminal phosphate group is modified to the RNA fragment split from the first strand (267nt), and the RNA fragment split from the second strand (220nt) is not modified.
  • RNA short chains were mixed in an equal molar ratio and assembled, and the synthesis conditions of the assembly were the same as those in Example 1.
  • the 9 junction sites in strand 1 were connected by T4 RNA ligase, so that all short chains in strand 1 formed a complete 267nt chain respectively.
  • the R-n series of short chains used in this experiment are all modified with a 5'-terminal phosphate group (except R-n15 and its corresponding base-modified short chains), and the R-m and R-y series of short chains are not modified.
  • RNA267/220 assembly was denatured to obtain a continuous first strand including a hairpin structure, and the results were observed by polyacrylamide gel electrophoresis. The results are shown in FIG. 8 .
  • RNA single strands up to 267 nt can be prepared in one pot.
  • the specific sequences of the short chains used are shown in Table 3.
  • Example 5 In order to prove the sequence correctness of the synthesized 267nt single chain, the obtained sequence was sequenced in Example 5 at the same time. The results are shown in Table 4. The effective signal regions were all aligned with the expected sequence, and there were no mismatches and gaps/inserts.
  • Sequencing direction number of bases detected Compare base regions Number of mismatches Number of gaps/inserts positive 241 40-264 0 0 reverse 244 2-231 0 0
  • RNA double strands up to 267/220 bp were split, and the method of splitting short strands was basically the same as that described in Example 5.
  • any one of the three short chains of R-n15, R-n16 or R-n17 is introduced into an m 6 A modification (the three RNA sequences containing the modification are called R-n15-m 6 A, R-n16- m 6 A and R-n17-m 6 A), or introduce a BrU modification into any of the four short chains of R-n15, R-n17, R-n20 or R-n21 (the four RNA sequences containing the modification are respectively referred to as R-n15-BrU, R-n17-BrU, R-n20-BrU, R-n21-BrU).
  • the 18 short chains described in Example 5 were mixed and assembled in an equimolar ratio, wherein the non-modified short chains R-n15, R-n16 or R-n17 were respectively modified short chains R-n15-m 6 A, R -n16-m 6 A and R-n17-m 6 A are substituted, and the unmodified short chains R-n15, R-n17, R-n20 or R-n21 are respectively modified short chains R-n15-BrU, R-n17 -BrU, R-n20-BrU, R-n21-BrU substitutions, each group only replaces one sequence, a total of 7 groups are assembled.
  • the synthesis conditions of the assembly were the same as in Example 1.
  • RNA267/220 assembly aqueous solutions with different site modifications After obtaining 7 sets of RNA267/220 assembly aqueous solutions with different site modifications, the 9 junction sites in chain 1 were connected by T4 RNA ligase, so that all short chains in chain 1 formed complete, banded 267nt chain with precise site modification.
  • the obtained linked chain 1 was observed by polyacrylamide gel electrophoresis, and the results are shown in FIG. 9 .

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Abstract

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

Description

一种制备定点修饰的长链RNA的方法 技术领域
本公开属于分子生物学和合成生物学领域，具体来说，本公开涉及一种制备长链RNA的方法及其所制备的长链RNA。
背景技术
近年来，基于mRNA的免疫疗法逐渐在遗传性代谢疾病 [1,2]、肿瘤治疗 [3-5]、抗病毒治疗 [6,7]及再生医学等生物医药领域展现出了令人瞩目的应用潜力，已有若干种基于mRNA的抗肿瘤药物得到了报道。此外，siRNA、miRNA、长链非编码RNA(Lnc RNA)、RNA适配体及核酶等RNA技术也在飞速发展，RNA在生物学上越来越受到重视。随着RNA在生物研究、药物研发以及临床中应用的不断推广，对RNA合成的质量也提出了更高的要求。
目前，非修饰RNA在生物医药领域展现出了丰富的应用前景。然而其在生物体系内的低稳定性极大地限制了非修饰RNA的实际治疗应用。研究显示，化学修饰(如碱基修饰 [8]、核糖修饰 [9]与磷酸骨架修饰 [10]等)的引入能够抑制RNA被核酸酶及免疫体系识别，从而提高其在生物体内的稳定性、降低免疫原性，使其展现出更加优异的疗效 [6,11]。
目前，RNA合成的方法包括固相合成法、体外转录、滚环转录(rolling circle transcription，RCT)等方法。其中，固相合成法(亚磷酰胺法)是目前应用最为广泛的合成方法，通常能够规模化合成60nt及以下的短链RNA [12]，且适用于对RNA中任意位点的精准修饰。然而，固相合成法在合成序列长度较长的目标RNA时，其合成产率随序列长度上升而呈指数下降，同时错误率迅速升高，因此尽管已有通过RNA固相合成法合成110nt RNA序列的报道 [13]，但目前利用化学合成法合成的、具有一定产率的RNA依然以60nt左右为普遍长度 [12]；目前已商品化的单链RNA合成最长能够达到120nt，但对于含有序列内部(即除5’与3’末端两个碱基之外的其他碱基上)定点修饰的RNA序列，则至多只能达到60nt。
对于更长的RNA，目前主要通过体外转录法实现其制备。基于T7、T3或SP6 RNA聚合酶等转录带有启动子的双链DNA模板获得长单链RNA，可获得大量的RNA产物，且转录长度不受限制，适于长链RNA的合成 [14]。例如，Bieker等 [15]最早于1984年基于RNA polymerase III实现了5S DNA的转录。但体外转录法对RNA聚合酶具有高度依赖性，且仅能通过酶的工程化 [16]、添加一定比例的修饰碱基单体 [8]等手段实现序列中的修饰碱基插入。但这种在体外转录过程中添加修饰碱基的方法由于酶对位点的非选择性，无法实现对特定位点进行的精准修饰，而只能实现整体修饰比例的控制，由此获得的含有修饰碱基的RNA序列在分子层面是一种由多种序列构成的混合物，这对其在生物医学中的应用特定位点化学修饰作用的深入研究造成了阻碍。RCT法同样能够实现长链RNA的合成，但此方法一般用于合成连续重复序列 [17]，其应用范围更加受限，且同样存在无法实现序列中修饰碱基的定点插入的问题 [18]。因此，目前亟需发展新的RNA合成方法，能够实现长链RNA的稳定、规模化生产，并且满足对RNA中特定修饰碱基的精准插入需求。
通过检索，我们在最近公开的专利文献中发现，专利CN103993002A中发明了一种大规模合成长链RNA药物的生产新工艺，其具体步骤包括：第一步，设计双链DNA转录模板；第二步，模板制备；第三步，模板纯化；第四步，体外转录；第五步，RNA产物纯化；第六步，纯化后RNA纯度检测。上述工艺虽然实现了大规模合成长链RNA药物，但是仍然属于传统分子生物学过程，即：基于DNA模板，利用RNA聚合酶在体外 进行转录，经纯化后获得30-200nt RNA单链。此专利中描述的方法未涉及含有非天然修饰碱基的RNA的制备，并且由前段所述原理可知，该方法不能实现在特定位点含有修饰碱基的RNA长链的制备。
引用文献：
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发明内容
发明要解决的问题
鉴于现有技术水平的限制，目前难以实现在特定位点含有修饰基团的长链RNA的合成。
在一些实施方式中，本公开提供了一种制备长链RNA的方法，基于精确组装与连接过程，能够在60nt以上，特别是60-1000nt范围内合成具有任意序列的长链RNA。
在另一些实施方式中，本公开的方法可用于制备任意位点可精准修饰的长链RNA，克服了目前无法合成位点精准修饰的长链RNA的技术障碍。
在另一些实施方式中，本公开的方法可用于制备单链的长链RNA，且所制备的单链的长链RNA可以在任意位点含有修饰基团，具有合成难度低、准确度高、成本低的优势。
用于解决问题的方案
本公开提供了一种制备长链RNA的方法，其包括以下步骤：
合成步骤：合成第一链的核酸片段组和第二链的核酸片段组，所述第一链的核酸片段组由RNA片段组成，所述第二链的核酸片段组由RNA片段和DNA片段中的至少一种组成；
所述第一链的核酸片段组包括RNA片段组a和任选的RNA片段组b，所述第二链的核酸片段组包括核酸片段组c和任选的核酸片段组d；所述RNA片段组a包括RNA片段n i和RNA片段n i+1，所述核酸片段组b包括RNA片段x ii和RNA片段x ii+1，所述核酸片段组c包括核酸片段m i，所述核酸片段组d包括核酸片段y ii和核酸片段y ii+1中的至少一种，i、ii彼此独立的选自1以上的整数；
其中，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列，核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列；RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构，所述5’延伸臂序列与核酸片段y ii的5’端序列为互补序列，所述3’延伸臂序列与核酸片段y ii+1的3’端序列为互补序列；
退火步骤：将所述第一链的核酸片段组和第二链的核酸片段组混合于同一反应体系中，退火，形成双链组装体前体；其中，所述第一链中相邻的两个核酸片段之间存在连接口，所述第二链中相邻的两个核酸片段之间存在连接口；所述第一链的核酸片段组中的相邻核酸片段之间的连接口与所述第二链的核酸片段组中的相邻核酸片段之间的连接口相互错开；
连接步骤：连接所述第一链的连接口，得到由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，所述第二链的核酸片段组由RNA片段组成。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，所述第二链的核酸片段组由DNA片段组成。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，所述第二链的核酸片段组由RNA片段和DNA片段组成。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述方法还包括如下步骤：
变性步骤：对所述双链组装体进行变性处理，得到连续化的单链RNA；
可选地，所述方法还包括纯化步骤：从所述反应体系中纯化所述连续化的单链RNA。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述发夹结构还包括形成双链结构的茎干区和未形成双链结构的茎环区，所述茎干区的5’末端和3’末端分别连接所述5’延伸臂和所述3’延伸臂；优选的，所述RNA片段x ii和RNA片段x ii+1之间的连接口位于所述茎干区。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，
所述RNA片段n i+1的3’端序列与所述核酸片段组c的其它核酸片段的3’端序列为互补序列或为未配对序列；或者，
所述核酸片段y ii的3’端序列与所述RNA片段组a的其它核酸片段的3’端序列为互补序列或为未配对序列；或者，
所述核酸片段y ii+1的5’端序列与所述RNA片段组a的其它核酸片段的5’端序列为互补序列或为未配对序列；
可选地，所述RNA片段n i+1的3’端序列与核酸片段m i+1的3’端序列为互补序列，所述核酸片段m i+1的5’端序列与所述核酸片段组a的其它核酸片段为互补序列或为未配对序列。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述连续化的单链RNA的长度为60nt以上，优选80nt以上，优选100nt以上，优选120nt以上，优选60-1000nt，优选80-600nt，更优选100-400nt，最优选120-360nt。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的长度为8-120nt，优选10-80nt，更优选15-40nt，最优选20-30nt。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的5’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt；或者，
所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的3’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述第一链的核酸片段组中任一核酸片段包含位于5’末端的磷酸基团，和位于3’末端的羟基；所述连接步骤中，将所述连接口两侧的磷酸基团和羟基连接为磷酸二酯键；
可选地，以酶连接或化学连接将所述相邻的磷酸基团和羟基连接为磷酸二酯键。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的碱基，且紧邻所述连接口的位置处的碱基为未修饰的碱基；
可选地，所述修饰选自m 6A、Ψ、m 1A、m 5A、ms 2i 6A、i 6A、m 3C、m 5C、ac 4C、m 7G、m2,2G、m 2G、m 1G、Q、m 5U、mcm 5U、ncm 5U、ncm 5Um、D、mcm 5s 2U、Inosine(I)、hm 5C、s 4U、s 2U、偶氮苯、Cm、Um、Gm、t 6A、yW、ms 2t 6A或其衍生物。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的核糖，且紧邻所述连接口的位置处的核糖为未修饰的核糖；
可选地，所述修饰选自LNA、2’-OMe、3’-OMeU、vmoe、2'-F或2’-OBn(2’-O-benzyl group)或其衍生物。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述第一链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的磷酸二酯键，且紧邻所述连接口的位置处的磷酸二酯键为未修饰的磷酸二酯键；
或者，所述第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的磷酸二酯键，且紧邻所述连接口的位置处的磷酸二酯键为未修饰的磷酸二酯键；
可选地，所述修饰选自phosphorothioate(PS)、nucleotide triphosphate(NTPαS)或其衍生物等。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述退火步骤中，将所述第一链的核酸片段组和所述第二链的核酸片段组孵育后，降温，形成双链组装体前体；
可选地，所述孵育的温度为0-100℃的任意温度，优选10-85℃的任意温度，更优选20-65℃区间内的任意温度，孵育时间为任意所需时间；
所述降温的速度为任意速度，降温至使反应体系中的核酸片段杂交形成双链组装体前体的任意温度即可。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述退火步骤中，将所述第一链的核酸片段组和第二链的核酸片段组溶解于同一溶剂中，得到所述反应体系。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述反应体系的pH为3-11，优选pH 4-10，更优选pH 5-9，最优选pH 6-8。
在一些实施方式中，根据本公开所述的制备长链RNA的方法，其中，所述反应体系中，所述第一链的核酸片段组和第二链的核酸片段组中任意两个核酸片段的摩尔比为1：(0.1-10)，优选1：(0.5-2)，最优选1：1。
本公开还提供了一种长链RNA，其中，所述长链RNA由根据本公开所述的方法制得，所述长链RNA为单链的长链RNA；
优选地，所述长链RNA的一个或多个位置处包含修饰的碱基、核糖或磷酸二酯键。
发明的效果
在一些实施方式中，本公开提供的制备长链RNA的方法，能够制得任意序列的长链RNA，且本发明所描述的方法不需要以DNA为模板、不依赖RNA聚合酶等，具有成本低、合成难度低、产率高、序列准确度高、可精准引入修饰等优势，适于大规模的生产应用。
在一些实施方式中，本公开提供的制备长链RNA的方法，能够制备得到具有发夹结构的长链RNA，以模拟RNA分子的天然空间构象，一方面提高了长链RNA的结构稳定性，另一方面有利于实现长链RNA的生物学功能。
在一些实施方式中，本公开提供的制备长链RNA的方法，通过制备由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体，双链组装体仅需通过简单的变性处理即能得到目标的长链RNA，对于非目标的单链核酸链在制备完成后即以核酸片段分散于反应体系中，不需要对其进行剪切或效果等的再处理，有效简化了单链的长链RNA的制备步骤，提高了制备效率。
在一些实施方式中，本公开提供的制备长链RNA的方法，在其中实现任意位点修饰碱基的精准插入，解决了目前长链RNA的合成方法中无法实现对特定位点进行精准修饰的问题。
在一些实施方式中，本公开提供的长链RNA，以上述制备长链RNA的方法制得，其序列准确度高，并可实现任意位点修饰碱基的精准插入，所获得的长链RNA及含有修饰碱基的长链RNA，在药物研发、临床治疗等方面具有广泛的应用前景。
附图说明
图1示出了长链RNA的组装示意图；
图2示出了长链RNA中发夹结构的组装示意图；
图3示出了包含发夹结构的长链RNA的组装示意图；
图4示出了RNA80/100组装体的非变性聚丙烯酰胺凝胶电泳表征结果；
图5示出了80nt RNA单链的变性聚丙烯酰胺凝胶电泳表征结果；
图6示出了RNA100/100、RNA140/120及RNA200/180三种双链RNA组装体的非变性聚丙烯酰胺凝胶电泳表征结果；
图7示出了100nt、140nt和200nt RNA单链的变性聚丙烯酰胺凝胶电泳表征结果；
图8示出了长达267nt RNA单链的变性聚丙烯酰胺凝胶电泳表征结果；
图9示出了含有定点修饰的267nt RNA单链的变性聚丙烯酰胺凝胶电泳表征结果。
具体实施方式
以下，针对本公开的内容进行详细说明。以下所记载的技术特征的说明基于本公开的代表性的实施方案、具体例子而进行，但本公开不限定于这些实施方案、具体例子。需要说明的是：
本公开中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。
本公开中，如没有特殊声明，则“多”、“多种”、“多个”等中的“多”表示2或以上的数值。
本公开中，所述“基本上”、“大体上”或“实质上”表示于相关的完美标准或理论标准相比，误差在5％以下，或3％以下或1％以下。
本公开中，如没有特别说明，则“％”均表示质量百分含量。
本公开中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
本公开中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。
本公开中，虽然所公开的内容支持术语“或”、“或者”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”、“或者”是指“和/或”。
本公开中所使用的“水”包括自来水、去离子水、蒸馏水、双蒸水、纯净水、离子交换水等任何可行的水。
在本公开中，“双链RNA的组装体”和“双链RNA”所代表的含义相同，可以互相替换。
在本公开中，“双链组装体”和“双链组装体前体”可以是由连续化的单链RNA和片段化的单链RNA互补形成，也可以由连续化的单链RNA和片段化的单链DNA互补形成，也可以由连续化的单链RNA和由RNA片段和DNA片段共同形成的片段化的单链核酸链互补形成。也就是说，在本公开中，“双链组装体”和“双链组装体前体”中的双链，不是DNA双链。
在本公开中，“连接口”又称缺口(nick)，其存在于单链核酸链的相邻的两个核苷酸之间，是由于相邻的两个核苷酸之间未形成磷酸二酯键而产生。
第一方面
本公开的第一方面提供了制备长链RNA的方法，其包括以下步骤：
合成步骤：合成第一链的核酸片段组和第二链的核酸片段组，所述第一链的核酸片段组由RNA片段组成，所述第二链的核酸片段组由RNA片段和DNA片段中的至少一种组 成；
所述第一链的核酸片段组包括RNA片段组a和任选的RNA片段组b，所述第二链的核酸片段组包括核酸片段组c和任选的核酸片段组d；所述RNA片段组a包括RNA片段n i和RNA片段n i+1，所述核酸片段组b包括RNA片段x ii和RNA片段x ii+1，所述核酸片段组c包括核酸片段m i，所述核酸片段组d包括核酸片段y ii和核酸片段y ii+1中的至少一种，i、ii彼此独立的选自1以上的整数；
其中，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列，核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列；RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构，所述5’延伸臂序列与核酸片段y ii的5’端序列为互补序列，所述3’延伸臂序列与核酸片段y ii+1的3’端序列为互补序列；
退火步骤：将所述第一链的核酸片段组和第二链的核酸片段组混合于同一反应体系中，退火，形成双链组装体前体；其中，所述第一链中相邻的两个核酸片段之间存在连接口，所述第二链中相邻的两个核酸片段之间存在连接口；所述第一链的核酸片段组中的相邻核酸片段之间的连接口与所述第二链的核酸片段组中的相邻核酸片段之间的连接口相互错开；
连接步骤：连接所述第一链的连接口，得到由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体。
需要说明的是，在目前的RNA合成方法中，通过短片段退火、连接口连接的方法一般仅用于短的双链RNA的合成。在专利文献CN102876658A中公开了一种大规模合成长链核酸分子的方法，在短链核酸分子片段的连接步骤中，将步骤1合成得到的包括上述纯化序列的小片段核酸分子在溶液中通过互补配对，形成带有每条单链均具有缺口的双链，在核酸分子连接酶的作用下，对两条链上的缺口进行连接，形成长双链组装体，通过PCR步骤，对大量混合产物中的长双链组装体进行扩增与纯化，最终获得由两条连续化单链互补形成的长双链核酸分子。
然而，本公开在研究中发现，上述长双链核酸分子的制备方法至少存在以下问题：(1)以上述公开的方法得到的是由两条连续化长链形成的双链结构的核酸分子，双链结构变性后在反应体系中得到均匀混合的两种连续化的长链，因此，当需要制备单链的长链核酸分子时，仅通过对双链结构的解链是无法实现的。事实上，对于长达60bp的双链分子，由于其解链温度极高，解链过程较为困难，因此单链的长链核酸分子的制备，也难以通过对双链的长链核酸分子进行解链来实现。(2)上述公开的方法未考虑核酸分子的天然构象，对于易形成二级结构的RNA分子，难以维持其天然的空间构象，难以保持其生物学功能。(3)RNA双链无法直接通过PCR进行扩增，因此上述公开方法无法直接用于RNA的制备。(4)上述公开方法中，必须通过PCR过程实现混合粗产物中少量的目标长双链核酸分子的扩增，而通过PCR过程，无法实现终产物中精准定点修饰的引入。
本公开的制备方法，将长链RNA划分为若干短的RNA片段，大大降低了长链RNA的合成难度。在合成长链RNA的过程中不需要以DNA为模板或使用RNA聚合酶等，部分情况下甚至能够以目标序列自身为模板，而无需额外加入RNA模板短链组，有效降低了长链RNA的化学合成难度，并且能够实现对长链RNA中任意位点的碱基、核糖或磷酸二酯键的修饰，规避了常规体外转录法难以实现定点修饰的问题，具有成本低、产率高、序列准确度高的优势。
此外，本公开的制备方法仅对由RNA片段形成的第一链的连接口进行连接，得到由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体，对于片段化的单链核酸链可以是由DNA片段和RNA片段中的至少一种组成。该制备方法中也不包括扩增步骤，且避免对双链组装体中片段化的单链核酸链(也即，单链RNA)的连接口进行连接，上 述的双链组装体仅通过简单的变性处理，即可实现对目标长链RNA的回收，制备方法中不包括对非目标的单链核酸链的消化、剪切等步骤，有效提高了单链的长链RNA的制备效率，适于大规模的工业化应用。
<划分长链RNA的序列>
在制备长链RNA之前，首先要对长链RNA的序列进行划分，附图1示出了一种长链的双链RNA结构，其中的第一链为目标合成的长链RNA，第二链为与第一链互补的单链核酸链。分别对第一链和第二链的核苷酸序列进行划分，使第一链和第二链的核苷酸序列被划分为若干短链的核酸片段序列。具体的，形成第一链的核酸片段组由RNA片段组成，形成第二链的核酸片段组由RNA片段和DNA片段中的至少一种组成。
进一步地，第一链的核酸片段组选自RNA片段组a和任选的RNA片段组b，第二链的核酸片段组选自核酸片段组c和任选的核酸片段组d。
对于第一链的核酸片段组，可以是RNA片段组a，或者是RNA片段组a和RNA片段组b的组合；对于第二链的核酸片段组，可以是核酸片段组c，或者是核酸片段组c和核酸片段组d的组合。
在一些实施方式中，核酸片段组c由RNA片段组成，核酸片段组d由RNA片段组成。在一些实施方式中，核酸片段组c由DNA片段组成，核酸片段组d由DNA片段组成。在一些实施方式中，核酸片段组c由RNA片段和DNA片段组成，核酸片段组d由RNA片段和DNA片段组成。在一些实施方式中，核酸片段组c由RNA片段和DNA片段组成，核酸片段组d由RNA片段组成。在一些实施方式中，核酸片段组c由RNA片段组成，核酸片段组d由RNA片段和DNA片段组成。在一些实施方式中，核酸片段组c由RNA片段和DNA片段组成，核酸片段组d由DNA片段组成。在一些实施方式中，核酸片段组c由DNA片段组成，核酸片段组d由RNA片段和DNA片段组成。
其中，RNA片段组a包括RNA片段n i和RNA片段n i+1，核酸片段组c包括核酸片段m i，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列，核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列。
如附图2和附图3所示，RNA片段组b包括RNA片段x ii和RNA片段x ii+1，核酸片段组d包括核酸片段y ii、核酸片段y ii+1或者同时包括核酸片段y ii和核酸片段y ii+1。其中，RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构，所述5’延伸臂序列与核酸片段y ii的5’端序列为互补序列，所述3’延伸臂序列与核酸片段y ii+1的3’端序列为互补序列。
在一些实施方式中，i、ii彼此独立的选自1以上的整数。
在一些实施方式中，第一链的RNA片段组为RNA片段组a，第二链的RNA片段组为RNA片段组c，也即，第一链的RNA序列被划分RNA片段n i的序列和RNA片段n i+1的序列，与第一链互补的第二链的核酸序列被划分为核酸片段m i的序列形成，通过核酸片段m i的5’端序列与核酸片段n i+1的5’端序列的互补配对，核酸片段m i的3’端序列与核酸片段n i的3’端序列的互补配对，实现对包含目标长链RNA(第一链)的双链RNA的序列划分。核酸片段m i可以是DNA片段或是RNA片段，均能实现对目标长链RNA的序列合成。
进一步的，RNA片段组a还可以包含其他的核酸片段。在一些实施方式中，RNA片段组a包括RNA片段n i、RNA片段n i+1、RNA片段n i+2。在一些实施方式中，RNA片段组a包括RNA片段n i、RNA片段n i+1、RNA片段n i+2、RNA片段n i+3。在一些实施方式中，RNA片段组a包括RNA片段n i、RNA片段n i+1、RNA片段n i+2、RNA片段n i+3、RNA片段n i+4。以此类推，RNA片段组a还可以包括其他数量的RNA片段，本公开对此不进行穷举。
进一步的，核酸片段组c还可以包含其他的核酸片段。在一些实施方式中，核酸片段组c包括核酸片段m i、核酸片段m i+1，其中核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列；核酸片段m i+1的3’端序列与RNA片段n i+1的3’端序列为互补序列，核酸片段m i+1的5’端序列为与RNA片段n i+2的5’端序列为互补序列或为未配对序列。其中，核酸片段m i、核酸片段m i+1彼此独立地为DNA片段或RNA片段。
在一些实施方式中，核酸片段组c包括核酸片段m i、核酸片段m i+1、核酸片段m i+2。其中核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列；核酸片段m i+1的3’端序列与RNA片段n i+1的3’端序列为互补序列，核酸片段m i+1的5’端序列为与RNA片段n i+2的5’端序列为互补序列；核酸片段m i+2的3’端序列与RNA片段n i+2的3’端序列为互补序列，核酸片段m i+2的5’端序列为与RNA片段n i+3的5’端序列为互补序列或为未配对序列。其中，核酸片段m i、核酸片段m i+1、核酸片段m i+2彼此独立地为DNA片段或RNA片段。
在一些实施方式中，核酸片段组c包括核酸片段m i、核酸片段m i+1、核酸片段m i+3。其中核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列；核酸片段m i+1的3’端序列与RNA片段n i+1的3’端序列为互补序列，核酸片段m i+1的5’端序列为与RNA片段n i+2的5’端序列为互补序列；核酸片段m i+2的3’端序列与RNA片段n i+2的3’端序列为互补序列，核酸片段m i+2的5’端序列为与RNA片段n i+3的5’端序列为互补序列；核酸片段m i+3的3’端序列与RNA片段n i+3的3’端序列为互补序列，核酸片段m i+3的5’端序列为与RNA片段n i+4的5’端序列为互补序列或为未配对序列。其中，核酸片段m i、核酸片段m i+1、核酸片段m i+2、核酸片段m i+3彼此独立地为DNA片段或RNA片段。以此类推，核酸片段组c还可以包括其他数量的核酸片段，本公开对此不进行穷举。
在一些实施方式中，第一链的RNA片段组为RNA片段组a和RNA片段组b，第二链的核酸片段组为核酸片段组c和核酸片段组d，也即，第一链的RNA序列(也即，目标长链RNA的序列)被划分为RNA片段n i的序列、RNA片段n i+1的序列、RNA片段x ii的序列和RNA片段x ii+1的序列，RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构；第二链的核酸序列被划分为核酸片段m i的序列、核酸片段y ii的序列和核酸片段y ii+1的序列，通过核酸片段m i的5’端序列与RNA片段n i+1的5’端序列的互补配对，核酸片段m i的3’端序列与RNA片段n i的3’端序列的互补配对，核酸片段y ii的5’端序列与5’延伸臂序列的互补配对，核酸片段y ii+1的3’端序列与3’延伸臂序列的互补配对，实现对包含发夹结构的目标长链RNA的双链RNA的序列划分。其中，核酸片段y ii、核酸片段y ii+1彼此独立地选自DNA片段或RNA片段，均能实现对目标长链RNA的序列合成。
进一步的，由RNA片段x ii和RNA片段x ii+1的部分碱基互补所形成的发夹结构还包括形成双链结构的茎干区和未形成双链结构的茎环区，所述茎干区的5’末端和3’末端分别连接所述5’延伸臂和所述3’延伸臂。对于第一链中是否需要设置包括RNA片段x ii和RNA片段x ii+1的核酸片段组b，可以根据目标的长链RNA的空间结构进行选择。例如，在进行核酸片段的序列划分之前，首先对目标长链RNA进行空间结构预测，若目标长链RNA中包含一个或多个发夹结构，将对应目标长链RNA中发夹结构的序列划分为RNA片段x ii和RNA片段x ii+1，将对应目标长链RNA中线性结构的序列划分为RNA片段n i和RNA片段n i+1。
进一步的，对与第一链互补的第二链进行序列划分，对应第一链的RNA片段n i和RNA片段n i+1划分核酸片段m i，对应第一链的发夹结构划分核酸片段y ii、核酸片段y ii+1 或核酸片段y ii与核酸片段y ii+1的组合。
在一些实施方式中，RNA片段组b包括RNA片段x ii和RNA片段x ii+1，由RNA片段x ii和RNA片段x ii+1形成第一链的第一发夹结构。在一些实施方式中，RNA片段组b包括RNA片段x ii、RNA片段x ii+1、RNA片段x ii+2、RNA片段x ii+3，其中，RNA片段x ii、RNA片段x ii+1形成第一链的第一发夹结构，RNA片段x ii+2、RNA片段x ii+3形成第一链的第二发夹结构。在一些实施方式中，RNA片段组b包括RNA片段x ii、RNA片段x ii+1、RNA片段x ii+2、RNA片段x ii+3、RNA片段x ii+4、RNA片段x ii+5，其中，RNA片段x ii、RNA片段x ii+1形成第一链的第一发夹结构，RNA片段x ii+2、RNA片段x ii+3形成第一链的第二发夹结构，RNA片段x ii+4、RNA片段x ii+5形成第一链的第三发夹结构。以此类推，RNA片段组b还可以包括其他数量的核酸片段，具体数量依据目标长链RNA中发夹结构的数量设置，本公开对此不进行穷举。
在一些实施方式中，核酸片段组d包括核酸片段y ii，核酸片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，核酸片段y ii的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列，核酸片段y ii为DNA片段或RNA片段。在一些实施方式中，核酸片段组d包括核酸片段y ii+1，核酸片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，核酸片段y ii+1的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列，核酸片段y ii+1为DNA片段或RNA片段。
在一些实施方式中，核酸片段组d包括核酸片段y ii和核酸片段y ii+1，核酸片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，核酸片段y ii的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，核酸片段y ii+1的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列。其中，核酸片段y ii、核酸片段y ii+1彼此独立地选自DNA片段或RNA片段。
在一些实施方式中，核酸片段组d包括核酸片段y ii、核酸片段y ii+1和核酸片段y ii+2，核酸片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，核酸片段y ii的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，核酸片段y ii+1的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+2的5’端序列与第二发夹结构的5’延伸臂序列互补，核酸片段y ii+2的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列。其中，核酸片段y ii、核酸片段y ii+1、核酸片段y ii+2彼此独立地选自DNA片段或RNA片段。
在一些实施方式中，核酸片段组d包括核酸片段y ii、核酸片段y ii+1和核酸片段y ii+3，核酸片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，核酸片段y ii的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，核酸片段y ii+1的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+3的3’端序列与第二发夹结构的3’延伸臂序列互补，核酸片段y ii+3的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列。其中，核酸片段y ii、核酸片段y ii+1、核酸片段y ii+3彼此独立地选自DNA片段或RNA片段。
在一些实施方式中，核酸片段组d包括核酸片段y ii、核酸片段y ii+1、核酸片段y ii+2和核酸片段y ii+3，核酸片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，核酸片段y ii的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，核酸片段y ii+1的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+2的5’端序列与第二 发夹结构的5’延伸臂序列互补，核酸片段y ii+2的3’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列；核酸片段y ii+3的3’端序列与第二发夹结构的3’延伸臂序列互补，核酸片段y ii+3的5’端序列与核酸片段组a中的核酸片段形成互补序列或为未配对序列。以此类推，核酸片段组b还可以包括其他数量的核酸片段，本公开对此不进行穷举。其中，核酸片段y ii、核酸片段y ii+1、核酸片段y ii+2、核酸片段y ii+3彼此独立地选自DNA片段或RNA片段。
具体而言，是否需要划分第一链的RNA片段组b及对应的第二链中的核酸片段组d由目标长链RNA的空间结构决定；RNA片段组b和核酸片段组d的实际数量由目标长链RNA中发夹结构的数量决定。而第一链中RNA片段组a的数量以及第二链中核酸片段组c的数量是由所需合成的目标长链RNA的序列决定的，通过对RNA片段组a和核酸片段组c中核酸片段数量的增加或减少，可以实现对不同长度的RNA链的划分，进而实现对所需长度和所需数量的长链RNA的合成。
在一些具体的实施方式中，核酸片段组a为RNA片段组a，核酸片段组c为RNA片段组c。RNA片段组a包括RNA片段n i和RNA片段n i+1，RNA片段组c包括RNA片段m i，RNA片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列，RNA片段m i的3’端序列与RNA片段n i的3’端序列为互补序列。
RNA片段组b包括RNA片段x ii和RNA片段x ii+1，RNA片段组d包括RNA片段y ii、RNA片段y ii+1或者同时包括RNA片段y ii和RNA片段y ii+1。其中，RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构，所述5’延伸臂序列与RNA片段y ii的5’端序列为互补序列，所述3’延伸臂序列与RNA片段y ii+1的3’端序列为互补序列。
在一些具体的实施方式中，第一链的核酸片段组为RNA片段组a，第二链的核酸片段组为RNA片段组c，也即，第一链的核酸序列被划分RNA片段n i的序列和RNA片段n i+1的序列，第二链的核酸序列被划分为RNA片段m i的序列形成，通过RNA片段m i的5’端序列与RNA片段n i+1的5’端序列的互补配对，RNA片段m i的3’端序列与RNA片段n i的3’端序列的互补配对，实现对包含目标长链RNA的双链RNA的序列划分。
进一步的，RNA片段组a还可以包含其他的RNA片段。在一些实施方式中，RNA片段组a包括RNA片段n i、RNA片段n i+1、RNA片段n i+2。在一些实施方式中，RNA片段组a包括RNA片段n i、RNA片段n i+1、RNA片段n i+2、RNA片段n i+3。在一些实施方式中，RNA片段组a包括RNA片段n i、RNA片段n i+1、RNA片段n i+2、RNA片段n i+3、RNA片段n i+4。以此类推，RNA片段组a还可以包括其他数量的RNA片段，本公开对此不进行穷举。
进一步的，RNA片段组c还可以包含其他的RNA片段。在一些实施方式中，RNA片段组c包括RNA片段m i、RNA片段m i+1，其中RNA片段m i的3’端序列与RNA片段n i的3’端序列为互补序列，RNA片段m i的5’端序列为与RNA片段n i+1的5’端序列为互补序列或为未配对序列；RNA片段m i+1的3’端序列与RNA片段n i+1的3’端序列为互补序列，RNA片段m i+1的5’端序列为与RNA片段n i+2的5’端序列为互补序列或为未配对序列。
在一些具体的实施方式中，RNA片段组c包括RNA片段m i、RNA片段m i+1、RNA片段m i+2。其中RNA片段m i的3’端序列与RNA片段n i的3’端序列为互补序列，RNA片段m i的5’端序列为与RNA片段n i+1的5’端序列为互补序列或为未配对序列；RNA片段m i+1的3’端序列与RNA片段n i+1的3’端序列为互补序列，RNA片段m i+1的5’端序列为与RNA片段n i+2的5’端序列为互补序列；RNA片段m i+2的3’端序列与RNA片段n i+2的3’端序列为互补序列，RNA片段m i+2的5’端序列为与RNA片段n i+3的5’端序列为互 补序列或为未配对序列。
在一些具体的实施方式中，RNA片段组c包括RNA片段m i、RNA片段m i+1、RNA片段m i+3。其中RNA片段m i的3’端序列与RNA片段n i的3’端序列为互补序列，RNA片段m i的5’端序列为与RNA片段n i+1的5’端序列为互补序列或为未配对序列；RNA片段m i+1的3’端序列与RNA片段n i+1的3’端序列为互补序列，RNA片段m i+1的5’端序列为与RNA片段n i+2的5’端序列为互补序列；RNA片段m i+2的3’端序列与RNA片段n i+2的3’端序列为互补序列，RNA片段m i+2的5’端序列为与RNA片段n i+3的5’端序列为互补序列；RNA片段m i+3的3’端序列与RNA片段n i+3的3’端序列为互补序列，RNA片段m i+3的5’端序列为与RNA片段n i+4的5’端序列为互补序列或为未配对序列。以此类推，RNA片段组c还可以包括其他数量的RNA片段，本公开对此不进行穷举。
在一些具体的实施方式中，第一链的核酸片段组为RNA片段组a和RNA片段组b，第二链的核酸片段组为RNA片段组c和RNA片段组d，也即，第一链的核酸序列被划分为RNA片段n i的序列、RNA片段n i+1的序列、RNA片段x ii的序列和RNA片段x ii+1的序列，RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构；第二链的核酸序列被划分为RNA片段m i的序列、RNA片段y ii的序列和RNA片段y ii+1的序列，通过RNA片段m i的5’端序列与RNA片段n i+1的5’端序列的互补配对，RNA片段m i的3’端序列与RNA片段n i的3’端序列的互补配对，RNA片段y ii的5’端序列与5’延伸臂序列的互补配对，RNA片段y ii+1的3’端序列与3’延伸臂序列的互补配对，实现对包含发夹结构的目标长链RNA的双链RNA的序列划分。
进一步的，由RNA片段x ii和RNA片段x ii+1的部分碱基互补所形成的发夹结构还包括形成双链结构的茎干区和未形成双链结构的茎环区，所述茎干区的5’末端和3’末端分别连接所述5’延伸臂和所述3’延伸臂。对于第一链中是否需要设置包括RNA片段x ii和RNA片段x ii+1的RNA片段组b，可以根据目标的长链RNA的空间结构进行选择。例如，在进行RNA片段的序列划分之前，首先对目标长链RNA进行空间结构预测，若目标长链RNA中包含一个或多个发夹结构，进而将目标长链RNA的序列设计为第一链，将对应目标长链RNA中发夹结构的序列划分为RNA片段x ii和RNA片段x ii+1，将对应目标长链RNA中线性结构的序列划分为RNA片段n i和RNA片段n i+1。
进一步的，对与第一链互补的第二链进行序列划分，对应第一链的RNA片段n i和n i+1划分m i，对应第一链的发夹结构划分RNA片段y ii、RNA片段y ii+1或RNA片段y ii与RNA片段y ii+1的组合。
在一些具体的实施方式中，RNA片段组b包括RNA片段x ii和RNA片段x ii+1，由RNA片段x ii和RNA片段x ii+1形成第一链的第一发夹结构。在一些实施方式中，RNA片段组b包括RNA片段x ii、RNA片段x ii+1、RNA片段x ii+2、RNA片段x ii+3，其中，RNA片段x ii、RNA片段x ii+1形成第一链的第一发夹结构，RNA片段x ii+2、RNA片段x ii+3形成第一链的第二发夹结构。在一些实施方式中，RNA片段组b包括RNA片段x ii、RNA片段x ii+1、RNA片段x ii+2、RNA片段x ii+3、RNA片段x ii+4、RNA片段x ii+5，其中，RNA片段x ii、RNA片段x ii+1形成第一链的第一发夹结构，RNA片段x ii+2、RNA片段x ii+3形成第一链的第二发夹结构，RNA片段x ii+4、RNA片段x ii+5形成第一链的第三发夹结构。以此类推，RNA片段组b还可以包括其他数量的RNA片段，具体数量依据目标长链RNA中发夹结构的数量设置，本公开对此不进行穷举。
在一些具体的实施方式中，RNA片段组d包括RNA片段y ii，RNA片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，RNA片段y ii的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列。在一些实施方式中，RNA片段组d包括RNA片段y ii+1，RNA片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，RNA片段 y ii+1的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列。
在一些具体的实施方式中，RNA片段组d包括RNA片段y ii和RNA片段y ii+1，RNA片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，RNA片段y ii的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，RNA片段y ii+1的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列。
在一些具体的实施方式中，RNA片段组d包括RNA片段y ii、RNA片段y ii+1和RNA片段y ii+2，RNA片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，RNA片段y ii的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，RNA片段y ii+1的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+2的5’端序列与第二发夹结构的5’延伸臂序列互补，RNA片段y ii+2的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列。
在一些具体的实施方式中，RNA片段组d包括RNA片段y ii、RNA片段y ii+1和RNA片段y ii+3，RNA片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，RNA片段y ii的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，RNA片段y ii+1的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+3的3’端序列与第二发夹结构的3’延伸臂序列互补，RNA片段y ii+3的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列。
在一些具体的实施方式中，组d包括y ii、y ii+1、y ii+2和y ii+3，RNA片段y ii的5’端序列与第一发夹结构的5’延伸臂序列互补，RNA片段y ii的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+1的3’端序列与第一发夹结构的3’延伸臂序列互补，RNA片段y ii+1的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+2的5’端序列与第二发夹结构的5’延伸臂序列互补，RNA片段y ii+2的3’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列；RNA片段y ii+3的3’端序列与第二发夹结构的3’延伸臂序列互补，RNA片段y ii+3的5’端序列与RNA片段组a中的RNA片段形成互补序列或为未配对序列。以此类推，RNA片段组b还可以包括其他数量的RNA片段，本公开对此不进行穷举。
在本公开中，5’端序列和3’端序列是指沿5’至3’的方向对核苷酸片段进行划分，使核苷酸片段被划分为两个区域。其中，将靠近5’末端的一个区域的序列称为5’端序列，将靠近3’末端的另一个区域的序列称为3’端序列。
在本公开中，5’末端是沿5’至3’的方向，位于核苷酸链中5’最尾端的位置处的一个核苷酸，其一般具有5’末端的磷酸基团。3’末端是沿5’至3’的方向，位于核苷酸链中3’最尾端的位置处的一个核苷酸，其一般具有3’末端的羟基。
进一步的，在第一链和第二链的核苷酸序列划分完成后，相连的两个核酸片段之间会存在连接口。例如，第一链中RNA片段n i和RNA片段n i+1之间存在连接口，第一链中RNA片段x ii和RNA片段x ii+1之间存在连接口，第二链中核酸片段m i和核酸片段m i+1之间存在连接口，核酸片段y ii和核酸片段y ii+1之间存在连接口。
为使退火后得到的双链组装体前体具有相对好的稳定性，对包含目标长链RNA的双链核酸链进行序列划分时，使得所述第一链的核酸片段组中的相邻核酸片段之间的连接口与所述第二链的核酸片段组中的相邻核酸片段之间的连接口相互错开。在一些优选的实施方式中，RNA片段x ii和RNA片段x ii+1之间的连接口位于发夹结构的茎干区。在此情况下，通过将存在二级结构的目标RNA序列划分为RNA片段x ii和RNA片段x ii+1， 利用目标RNA序列自身互补形成发夹结构，能够最大程度地保留连接完成后所形成的单链的长链RNA(尤其是具有特殊生理功能的RNA序列)的高级结构，从而确保其生理功能不受影响。
进一步的，对第一链和第二链进行序列划分时，应使第一链的核酸片段组和第二链的核酸片段组中的核酸片段的解链温度(T m)应尽可能接近，并且避免链内复杂高级结构的存在，以降低核酸片段退火形成双链组装体前体的难度。
在一些具体的实施方式中，第一链的核酸片段组和第二链的核酸片段组中任一核酸片段的5’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。例如，任一核酸片段的5’端序列的长度为4nt、6nt、8nt、10nt、12nt、14nt、16nt、18nt等等。
在一些更为具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段的5’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。例如，任一RNA片段的5’端序列的长度为4nt、6nt、8nt、10nt、12nt、14nt、16nt、18nt等等。
在一些更为具体的实施方式中，第二链的核酸片段组为RNA片段组、DNA片段组、或RNA片段和DNA片段的组合，其中任一RNA片段或任一DNA片段的5’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。例如，任一RNA片段的5’端序列的长度为4nt、6nt、8nt、10nt、12nt、14nt、16nt、18nt等等。
在一些具体的实施方式中，第一链的核酸片段组和第二链的核酸片段组中任一核酸片段的3’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。例如，任一核酸片段的3’端序列的长度为4nt、6nt、8nt、10nt、12nt、14nt、16nt、18nt等等。
在一些更为具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段的3’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。例如，任一RNA片段的3’端序列的长度为4nt、6nt、8nt、10nt、12nt、14nt、16nt、18nt等等。
在一些更为具体的实施方式中，第二链的核酸片段组为RNA片段组、DNA片段组、或RNA片段和DNA片段的组合，其中任一RNA片段或任一DNA片段的3’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。例如，任一RNA片段的3’端序列的长度为4nt、6nt、8nt、10nt、12nt、14nt、16nt、18nt等等。
在一些具体的实施方式中，所述连续化的单链RNA的长度为60-1000nt，优选80-600nt，更优选100-400nt，最优选120-360nt。例如，任一单链核酸链的长度为60nt、70nt、80nt、90nt、100nt、120nt、140nt、160nt、180nt、200nt、220nt、240nt、250nt、260nt、267nt、270nt、300nt、320nt、340nt、360nt、400nt、500nt、600nt、700nt、800nt、900nt、1000nt等等。
在一些具体的实施方式中，第一链为由RNA片段组装形成的单链RNA，将第一链中的连接口连接后，得到连续化的单链RNA，连续化的单链RNA的长度为60-1000nt，优选80-600nt，更优选100-400nt，最优选120-360nt。例如，单链RNA的长度为60nt、70nt、80nt、90nt、100nt、120nt、140nt、160nt、180nt、200nt、220nt、240nt、250nt、260nt、267nt、270nt、300nt、320nt、340nt、360nt、400nt、500nt、600nt、700nt、800nt、900nt、1000nt等等。
在一些具体的实施方式中，第二链为单链RNA、单链DNA、或DNA和RNA的混合单链，第二链存在于双链组装体中，为片段化的单链核酸链。双链组装体中第二链的长度为60-1000nt，优选80-600nt，更优选100-400nt，最优选120-360nt。例如，单链 RNA的长度为60nt、70nt、80nt、90nt、100nt、120nt、140nt、160nt、180nt、200nt、220nt、240nt、250nt、260nt、267nt、270nt、300nt、320nt、340nt、360nt、400nt、500nt、600nt、700nt、800nt、900nt、1000nt等等。
在一个具体的实施方式中，本公开记载了一种制备单链的长链RNA的方法，其包括以下步骤：
合成步骤：合成第一链的核酸片段组和第二链的核酸片段组，所述第一链的核酸片段组由RNA片段组成，所述第二链的核酸片段组由RNA片段和DNA片段中的至少一种组成；
所述第一链的核酸片段组包括RNA片段组a和任选的RNA片段组b，所述第二链的核酸片段组包括核酸片段组c和任选的核酸片段组d；所述RNA片段组a包括RNA片段n i和RNA片段n i+1，所述核酸片段组b包括RNA片段x ii和RNA片段x ii+1，所述核酸片段组c包括核酸片段m i，所述核酸片段组d包括核酸片段y ii和核酸片段y ii+1中的至少一种，i、ii彼此独立的选自1以上的整数；
其中，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列，核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列；RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构，所述5’延伸臂序列与核酸片段y ii的5’端序列为互补序列，所述3’延伸臂序列与核酸片段y ii+1的3’端序列为互补序列；
退火步骤：将所述第一链的核酸片段组和第二链的核酸片段组混合于同一反应体系中，退火，形成双链组装体前体；其中，所述第一链中相邻的两个核酸片段之间存在连接口，所述第二链中相邻的两个核酸片段之间存在连接口；所述第一链的核酸片段组中的相邻核酸片段之间的连接口与所述第二链的核酸片段组中的相邻核酸片段之间的连接口相互错开；
连接步骤：连接所述第一链的连接口，得到由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体。
变性步骤：对所述双链组装体进行变性处理，得到连续化的单链RNA。
<合成核酸片段>
在将目标长链RNA的序列划分完成后，合成所需序列和所述数量的核酸片段。核酸片段的合成方法可以采用本领域中常用的RNA、DNA合成方法，例如，固相合成。以固相合成法可大规模制备短链的核酸片段，并且保证核酸片段的序列准确性。
需要说明的是，目前的RNA合成方法中，能够实现特定位置修饰引入的固相合成法通常仅用于60nt以下短链RNA的合成，最长不超过120nt，且利用固相合成法制备长度在60-120nt范围内的短链RNA时已很难引入定点修饰；而适用于长链RNA合成的体外转录法及RCT法等则难以实现定点修饰的引入。因此，对于超过60nt的长链RNA的合成，通常难以实现在合成过程中对长链RNA中特定位置处的碱基、核糖或磷酸二酯键的精确修饰。由此可知，现有技术中对长度超过60nt，特别是超过120nt的长链RNA中任意位点的精确修饰尚存在技术障碍，这极大地限制了长链RNA在生物医药领域中的应用。
在一些具体的实施方式中，第一链的核酸片段组和第二链的核酸片段组中任一核酸片段的长度为8-120nt，优选10-80nt，更优选15-40nt，最优选20-30nt。例如，核酸片段的长度为22nt、24nt、26nt、28nt、30nt、40nt、50nt、60nt、70nt、80nt、90nt、100nt等等。核酸片段的长度决定了其合成的难度和成本，将核酸片段的长度控制在20-30nt，可以有效降低核酸片段的合成难度，控制合成成本。
在一些更为具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段的长度为8-120nt，优选10-80nt，更优选15-40nt，最优选20-30nt。 例如，RNA片段的长度为22nt、24nt、26nt、28nt、30nt、40nt、50nt、60nt、70nt、80nt、90nt、100nt等等。
在一些更为具体的实施方式中，第二链的核酸片段组为RNA片段组、DNA片段组或RNA片段和DNA片段的组合，其中任一RNA片段或任一DNA片段的长度为8-120nt，优选10-80nt，更优选15-40nt，最优选20-30nt。例如，RNA片段的长度为22nt、24nt、26nt、28nt、30nt、40nt、50nt、60nt、70nt、80nt、90nt、100nt等等。
在一些具体的实施方式中，第一链的核酸片段组和第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的碱基。例如，在核酸片段的1个、2个、3个、4个等等位置处包含修饰的碱基。碱基修饰的方法可以采用本领域中常用方法，例如，在化学合成短链核酸片段的过程中引入修饰的碱基。在合成核酸片段的过程中引入修饰的碱基，可实现对任意位点的碱基修饰，核酸片段装配为长链RNA后，能够得到可对任意位点的碱基进行精准修饰的长链RNA。
具体的，对于核酸片段中任意一个位置处碱基的修饰方式，可以选自m 6A、Ψ、m 1A、m 5A、ms 2i 6A、i 6A、m 3C、m 5C、ac 4C、m 7G、m2,2G、m 2G、m 1G、Q、m 5U、mcm 5U、ncm 5U、ncm 5Um、D、mcm 5s 2U、Inosine(I)、hm 5C、s 4U、s 2U、偶氮苯、Cm、Um、Gm、t 6A、yW、ms 2t 6A或其衍生物 [19,20]。
在一些更为具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段的一个或多个位置处包含修饰的核糖。例如，在RNA片段的1个、2个、3个、4个等等位置处包含修饰的碱基。
在一些具体的实施方式中，第一链的核酸片段组和第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的核糖。例如，在核酸片段的1个、2个、3个、4个等等位置处包含修饰的核糖。核糖修饰的方法可以采用本领域中常用方法，例如，在化学合成短链核酸片段的过程中引入修饰的核糖。在合成核酸片段的过程中引入修饰的核糖，可实现对任意位点的核糖修饰，核酸片段装配为长链RNA后，能够得到可对任意位点的核糖进行精准修饰的长链RNA。
具体的，对于核酸片段中任意一个位置处的核糖的修饰方式，可以选自LNA、2’-OMe、3’-OMeU、vmoe、2’-F或2’-OBn(2’-O-benzyl group)或其衍生物 [21]。
在一些更为具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段的一个或多个位置处包含修饰的核糖。例如，在RNA片段的1个、2个、3个、4个等等位置处包含修饰的核糖。
在一些具体的实施方式中，第一链的核酸片段组和第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的磷酸二酯键，磷酸二酯键形成于短链核酸片段的相邻的两个核苷酸之间。例如，在核酸片段的1个、2个、3个、4个等等位置处包含修饰的磷酸二酯键。磷酸二酯键修饰的方法可以采用本领域中常用方法，例如，在化学合成短链核酸片段的过程中引入修饰的磷酸二酯键。在合成核酸片段的过程中引入修饰的磷酸二酯键，可实现对任意位点的磷酸二酯键修饰，核酸片段装配为长链RNA后，能够得到可对任意位点的磷酸二酯键进行精准修饰的长链RNA。
具体的，对于核酸片段中任意一个位置处磷酸二酯键的修饰方式，可以选自Phosphorothioate(PS)、nucleotide triphosphate(NTPαS)或其衍生物 [22,23]。
在一些优选的实施方式中，对碱基、核糖和磷酸二酯键的修饰应该避开紧邻连接口位置处的碱基、核糖和磷酸二酯键，以避免第一链或第二链连接口处的修饰可能对后续双链RNA的组装体前体中的连接口连接造成影响。
在一些更为具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段的一个或多个位置处包含修饰的磷酸二酯键，磷酸二酯键形成于短链核 酸片段的相邻的两个核糖核苷酸之间。例如，在核酸片段的1个、2个、3个、4个等等位置处包含修饰的磷酸二酯键。
通过对核酸片段中任意一个或多个位点的碱基、核糖和磷酸二酯键中至少一者的修饰，将修饰后的核酸片段应用于本公开中长链RNA的合成中，能够实现对长链RNA中任意位点的精准修饰，有效解决了目前本领域中难以合成特定位点精准修饰的长链RNA的问题。修饰后的长链RNA不仅结构稳定性提高，还可进一步改善长链RNA的免疫原性等生物学性能，从而使合成的长链RNA在生物医学领域获得广泛的应用。
在一些具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段包含5’末端的磷酸基团，和3’末端的羟基。例如，RNA片段n i的5’末端包含磷酸基团，3’末端包含羟基；RNA片段n i+1的5’末端包含磷酸基团，3’末端包含羟基，RNA片段n i+2的5’末端包含磷酸基团，3’末端包含羟基；RNA片段n i+3的5’末端包含磷酸基团，3’末端包含羟基；RNA片段x ii的5’末端包含磷酸基团，3’末端包含羟基；当第一链和第二链的核酸片段组装形成具有发夹结构的双链组装体前体后，通过将连接口两侧的5’磷酸基团和3’羟基连接为磷酸二酯键，可以实现对第一链中连接口的连接，从而得到连续化的单链RNA(第一链)与片段化的单链核酸链(第二链)互补形成的双链组装体。
在一些具体的实施方式中，第一链的核酸片段组为RNA片段组，RNA片段组中任一RNA片段包含5’末端的磷酸基团，和3’末端的羟基。例如，RNA片段n i的5’末端包含磷酸基团，3’末端包含羟基；RNA片段n i+1的5’末端包含磷酸基团，3’末端包含羟基，RNA片段n i+2的5’末端包含磷酸基团，3’末端包含羟基；RNA片段n i+3的5’末端包含磷酸基团，3’末端包含羟基。当第一链和第二链的核酸片段组装形成具有发夹结构的双链RNA的组装体前体后，通过将连接口两侧的5’磷酸基团和3’羟基连接为磷酸二酯键，可以实现对第一链中连接口的连接，从而得到连续化的单链RNA(第一链)与片段化的单链核酸链(第二链)互补形成的双链组装体。
示例性的，RNA片段中5’末端的磷酸基团的引入方法可以采用本领域中常用的修饰方法，例如，可以在合成RNA片段的过程中直接在RNA片段的5’末端引入磷酸基团；或者是对未引入磷酸基团的RNA片段进行激酶处理，以对RNA片段的5’末端进行磷酸基团的修饰。
以上述的设计方法，进而第一链和第二链中的目标单链RNA的RNA片段中进行5’磷酸基团和3’羟基的添加，进而实现连接步骤中仅对目标单链RNA进行连接，得到连续化的单链RNA，而与目标单链RNA互补的核酸链仍为片段化的核酸链，有效避免了在后续回收目标单链RNA时需要对其互补链进行消化、剪切等处理。具体的，片段化的核酸链可以是由RNA片段组成的核酸链，由DNA片段组成的核酸链，或者是由RNA片段和DNA片段共同组成的核酸链。
<双链组装体前体>
将所述第一链的核酸片段组和第二链的核酸片段组混合于同一反应体系中，退火，得到由第一链和第二链至少部分互补形成的双链组装体前体；其中，所述第一链中相邻的两个核酸片段之间存在连接口，所述第二链中相邻的两个核酸片段之间存在连接口；所述第一链的核酸片段组中的相邻核酸片段之间的连接口与所述第二链的核酸片段组中的相邻核酸片段之间的连接口相互错开。
DNA分子含有4种脱氧核糖核苷酸，根据碱基种类的不同，分别为腺嘌呤脱氧核糖核苷酸(A)、鸟嘌呤脱氧核糖核苷酸(G)、胞嘧啶脱氧核糖核苷酸(C)和胸腺嘧啶脱氧核糖核苷酸(T)。与含有4种脱氧核糖核苷酸的DNA类似，RNA分子含有4种不同的核糖核苷酸，根据碱基种类的不同，分别为腺嘌呤核糖核苷酸(A)、鸟嘌呤核糖核 苷酸(G)、胞嘧啶核糖核苷酸(C)和尿嘧啶核糖核苷酸(U)。碱基与碱基之间能够通过氢键相互连接，其中，A与T、A与U、C与G之间分别能够形成氢键。碱基对之间精确的互补配对能力，使得序列彼此互补的两条反向核酸单链之间能够依靠氢键作用形成准确的双链结构。在制备双链组装体前体时，反应体系中的第一链的核酸片段和第二链的核酸片段经退火后能够在碱基互补配对原则的指导下重新组装为初始的目标长双链结构。
具体的，将第一链的核酸片段组和第二链的核酸片段组溶解于同一溶剂中，使两者充分混合，得到用于制备双链组装体前体的反应体系。本公开对具体的溶剂不作特别限定，可以是本领域常用的极性溶剂，例如：水等。对于反应体系中核酸片段混合的摩尔比，所述第一链的核酸片段组和第二链的核酸片段组中任意两个核酸片段的摩尔比为1：(0.1-10)，优选1：(0.5-2)，最优选1：1。示例性的，任意两个核酸片段的摩尔比为1:0.2、1:0.4、1:0.6、1:0.8、1:1、1:2、1:4、1:6、1:8等。通过设置核酸片段的摩尔比，可提高短链核酸片段的装配效率。
在一些具体的实施方式中，将第一链的RNA片段组和第二链的RNA片段组溶解于同一溶剂中，使两者充分混合，得到用于制备双链RNA的组装体前体的反应体系。本公开对具体的溶剂不作特别限定，可以是本领域常用的极性溶剂，例如：水等。对于反应体系中RNA片段混合的摩尔比，所述第一链的RNA片段组和第二链的RNA片段组中任意两个RNA片段的摩尔比为1：(0.1-10)，优选1：(0.5-2)，最优选1：1。示例性的，任意两个RNA片段的摩尔比为1:0.2、1:0.4、1:0.6、1:0.8、1:1、1:2、1:4、1:6、1:8等。通过设置RNA片段的摩尔比，可提高短链RNA片段的装配效率。
为进一步提高双链组装体前体的装配效率，提高双链RNA的组装体的产率，设置反应体系的pH为3-11，优选pH 4-10，更优选pH 5-9，最优选pH 6-8。示例性的，反应体系的pH为6、7、8、9等等。
进一步的，将所述第一链的核酸片段组和所述第二链的核酸片段组孵育后，降温，形成双链组装体前体；
可选地，所述孵育的温度为0-100℃的任意温度，优选10-85℃的任意温度，更优选20-65℃区间内的任意温度，孵育时间为任意所需时间。
所述降温的速度为任意速度，降温至使反应体系中的核酸片段杂交形成双链组装体前体的任意温度即可。
<双链组装体>
仅对存在于第一链的连接口进行连接，形成双链组装体。
具体的，是将连接口两侧的5’磷酸基团和3’羟基连接为磷酸二酯键。其中，连接方法可以是以T4 RNA连接酶进行连接的酶连接。或是化学连接的方法。连接口连接后得到第一链和第二链互补形成的完整的双链组装体，双链组装体中的第一链为连续化的单链RNA，第二链为RNA片段、DNA片段或RNA片段与DNA片段共同组成的片段化的单链核酸链，实现对长链RNA的制备。
进一步的，本公开的制备方法还包括变性步骤。对于变性步骤，是通过对所述双链组装体进行变性处理，得到连续化的单链RNA，即为目标的长链RNA。变性处理的方法可以是本领域中常用的将双链RNA解链形成单链RNA的方法。例如，在70℃的温度下处理5min，得到单链RNA。
进一步的，本公开的制备方法还包括纯化步骤。对于纯化步骤，是从所述反应体系中纯化所述连续化的单链RNA，本公开对纯化的方法不作具体限定，可以是从反应体系中高效回收RNA的各类方法。纯化步骤后得到的不含其它物质的长链RNA，可进一步应用于临床、药物研发、生物学研究等不同领域。
本公开中的制备方法在具有常规RNA化学合成法的所有优势(包括无需模板链、可精确定点修饰等)的同时，通过将目标长链RNA分割为若干段较短的RNA片段，而与目标的单链RNA互补的单链核酸链可分割为若干段较短的RNA片段、DNA片段，或是RNA片段与DNA片段的组合。通过这种序列的设计方式，大大降低了化学合成难度，并保留了化学合成法制备短链核酸片段时的高准确度、高产率与定点修饰能力。
将能够通过固相合成法轻松制得的核酸片段通过核酸自组装能力按特定顺序重新组合为目标结构的双链组装体前体，并以酶连或化学连接等技术将组装体中的连接口通过磷酸二酯键重新连接，获得由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体。对于双链组装体仅需要简单变性，即可得到单链的目标的长链RNA。由于固相合成过程能够实现初始短链核酸片段任意位点(除紧邻连接口两侧的碱基之外)的精准修饰，因此所获得的目标长链RNA也具有能够在几乎任意位点被准确修饰的特性。
第二方面
本公开的第二方面提供了一种长链RNA，长链RNA由第一方面的方法制得。可选地，所述长链RNA为单链的长链RNA。
本公开的长链RNA能够实现在任意位点的准确修饰，且长链RNA本身与修饰均无序列依赖性，为拓展长链RNA(尤其是具有精准修饰的长链RNA)在生物医学领域中的应用提供了基础。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
引用文献：
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实施例
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限定本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售获得的常规产品。
材料和方法
实施例所用的RNA序列均从睿博兴科公司购买，使用前未经额外处理。所有实验用水均采用18.2MΩcm密理博公司生产的超纯水。T4 RNA ligase 2及10X ligase buffer购买自NEB公司。其它化学试剂均采用分析纯。
实施例1. 100/80bp RNA双链组装体的构建
(1)以第一链与第二链分别长80nt与100nt的长链RNA(称为RNA80/100)作为目标长链，将长链切割为9条长为20nt的短链RNA。其中，R-n1、R-n2、R-n3和R-n4为合成第一链的RNA片段，R-m1、R-m2、R-m3、R-m4和R-m5为合成第二链的RNA片段。
(2)通过化学合成制备9条RNA片段。
所用的9条链的具体序列如下表所示：
表1.制备组装体所用的9条RNA短链的序列信息
序列名称 序列信息 nt SEQ ID NO
R-m1 AAAAGGAAAAGCGAUGCUAU 20 SEQ ID NO：1
R-n1 UACGAAUCCAAUAGCAUCGC 20 SEQ ID NO：2
R-m2 UGGAUUCGUAGGACUGCCUG 20 SEQ ID NO：3
R-n2 CAAGUAGUUACAGGCAGUCC 20 SEQ ID NO：4
R-m3 UAACUACUUGUCACUCUCUU 20 SEQ ID NO：5
R-n3 UGUCGGUAAGAAGAGAGUGA 20 SEQ ID NO：6
R-m4 CUUACCGACAAAACCUAAAU 20 SEQ ID NO：7
R-n4 UGAACAGAUAAUUUAGGUUU 20 SEQ ID NO：8
R-m5 UAUCUGUUCAAAAAGGAAAA 20 SEQ ID NO：9
(3)将上述9条RNA片段等摩尔比混合于1×TAE-Mg 2+缓冲液内，每条链浓度均为10μM，于70℃下加热5min，随后逐步冷却至室温，4℃下放置10min，得到目标RNA组装体。所得组装体以非变性聚丙烯酰胺凝胶电泳进行观察，结果如图4所示。
由图4可知，所述条件下以较高效率制得了目标双链RNA组装体前体。
实施例2. 80nt RNA单链的合成与凝胶电泳表征
(1)合成实施例1中9条RNA片段。
(2)对第一链(80nt)分割而成的R-n1、R-n2、R-n3和R-n4进行5’末端磷酸基团修饰，第二链分割而成的R-m1、R-m2、R-m3、R-m4和R-m5不进行修饰。其中，5’末端磷酸基团在合成R-n1、R-n2、R-n3和R-n4的过程中引入。
(3)按照实施例1中的方法获得RNA80/100组装体水溶液，其浓度为10μM。向10μL组装体水溶液内加入不同用量的T4 RNA ligase(10、20、40或80U)与2μL 10X T4 RNA ligase buffer，补充超纯水至总体积为20μL。体系在37℃下反应10min，以利用T4 RNA ligase对第一链中的3个连接口位点进行连接，使第一链中的4条短链形成一条完整的80nt链。
(4)向连接后的RNA80/100组装体中加入等体积甲酰胺，混匀后，在70℃下加热5min，立即用液氮冷却，以进行变性处理，得到连续化的第一链，所得80nt第一链以聚丙烯酰胺凝胶电泳进行观察，结果如图5所示。
由图5可知，所述条件下以较高效率制得了目标80nt RNA单链。
实施例3.更长RNA双链组装体的制备
为了合成更长的RNA序列，实施例3中将该体系进行了拓展，增加了短链RNA的数量。具体步骤如下：
(1)以第一链与第二链分别长200nt与180nt的长链RNA(称为RNA200/180)作为目标长链，将长链切割为19条长为20nt的短链RNA。从链1的5’末端开始，分别取其中的10条、13条及19条短链进行组装(分别称为RNA100/100、RNA140/120及RNA200/180)。其中，第一链(200nt)分割而成的RNA片段包括：R-n5、R-n6、R-n7、R-n8、R-n9、R-n10、R-n11、R-n12、R-n13、R-n14；第二链(180nt)分割而成的RNA片段包括：R-m6、R-m7、R-m8、R-m9、R-m10、R-m11、R-m12、R-m13、R-m14。
(2)以实施例1中所示的方法合成RNA组装体。
结果如图6所示，利用该方法，可以一锅内高效地组装出140/120bp的RNA长链，或通过分步组装得到200/180bp的RNA双链组装体，为后续长序列的合成提供了保障。
所用的19条链的具体序列如下表所示：
表2.制备组装体所用的19条RNA短链的序列信息
序列名称 序列信息 nt SEQ ID NO
R-n5 GUUCUUCUCUAGCUAUCCAU 20 SEQ ID NO：10
R-m6 CACUACCAGGAUGGAUAGCU 20 SEQ ID NO：11
R-n6 CCUGGUAGUGCAGCAUAAUC 20 SEQ ID NO：12
R-m7 GUAUCAGAGAGAUUAUGCUG 20 SEQ ID NO：13
R-n7 UCUCUGAUACGACAUGUAAG 20 SEQ ID NO：14
R-m8 AAAACCUCUUCUUACAUGUC 20 SEQ ID NO：15
R-n8 AAGAGGUUUUACAACUUGGA 20 SEQ ID NO：16
R-m9 GAAUACCAUGUCCAAGUUGU 20 SEQ ID NO：17
R-n9 CAUGGUAUUCAUUCGCUAAG 20 SEQ ID NO：18
R-m10 GUGUUGAAUGCUUAGCGAAU 20 SEQ ID NO：19
R-n10 CAUUCAACACCCAAUUUAUG 20 SEQ ID NO：20
R-m11 UAUCGUUGUGCAUAAAUUGG 20 SEQ ID NO：21
R-n11 CACAACGAUAGCGAUGCUAU 20 SEQ ID NO：22
R-m12 UACGAAUCCAAUAGCAUCGC 20 SEQ ID NO：23
R-n12 UGGAUUCGUAGGACUGCCUG 20 SEQ ID NO：24
R-m13 CAAGUAGUUACAGGCAGUCC 20 SEQ ID NO：25
R-n13 UAACUACUUGUCACUCUCUU 20 SEQ ID NO：26
R-m14 UGUCGGUAAGAAGAGAGUGA 20 SEQ ID NO：27
R-n14 CUUACCGACAAAACCUAAAU 20 SEQ ID NO：28
实施例4. 200nt RNA单链的制备
(1)在实施例3中所涉及的RNA100/100、RNA140/120及RNA200/180 3组目标RNA长链中，对第一链(100nt、140nt及200nt)分割而成的20ntRNA片段进行5’末端磷酸基团修饰，修饰方法同实施例2，对第二链(100nt、120nt及180nt)分割而成的RNA片段不进行修饰。
(2)按照实施例3中的方法获得RNA100/80组装体水溶液，随后通过T4 RNA ligase对链1中的4个、6个及9个连接口位点进行连接，连接方法同实施例2，使第一链中的所有RNA片段分别形成完整的100nt、140nt及200nt链。
(3)将步骤(2)中的RNA片段进行变性处理，变性处理方法同实施例2，得到连续化的第一链，第一链以聚丙烯酰胺凝胶电泳进行观察，结果如图7所示。
由图7可知，利用该方法，可以一锅内制备出不同长度的RNA单链，所示RNA单链长度分别为100、140及200nt。
实施例5. 267nt RNA单链的制备
为了合成功能性RNA序列，实施例5中对长达267/220bp的RNA双链进行了分割。具体如下：
(1)将长度为267nt的第一链分割为10条RNA片段，其长度为24-44nt不等，第一链的RNA片段包括表3中的R-n15、R-n16、R-n17、R-n18、R-x1、R-x2、R-n19、R-n20、R-n21、R-n22；将长度为220nt的第二链分割为8条短链，其长度为24-30nt不等，第二链的RNA片段包括表3中的R-m15、R-m16、R-y1、R-y2、R-m17、R-m18、R-m19、R-m20。需要说明的是，由于目标链1(第一链)中存在部分天然发夹结构，在设计组装体时，部分区域(R-x1及R-x2)利用链1的自组装发夹结构形成，而无需对应的互补链(链2)，因此链2与链1并非完全对应互补。
(2)在目标RNA长链中，对第一链(267nt)分割而成的RNA片段进行5’末端磷酸基团修饰，第二链(220nt)分割而成的RNA片段不修饰。
(3)将上述18条RNA短链等摩尔比混合并进行组装，组装体的合成条件与实施例1中相同。获得RNA267/220组装体水溶液后，通过T4 RNA ligase对链1中的9个连接口位点进行连接，使链1中的所有短链分别形成完整的267nt链。需要说明的是，本实验中所用R-n系列短链均带有5’末端磷酸基团修饰(R-n15及其对应的碱基修饰短链除外)，R-m与R-y系列短链均无修饰。
(4)对RNA267/220组装体进行变性处理，得到包含发夹结构的连续化的第一链，以聚丙烯酰胺凝胶电泳进行观察，结果如图8所示。
如图8所示，利用该方法，可以一锅内制备出长达267nt的RNA单链。所用短链的具体序列如表3所示。
为了证明所合成267nt单链的序列正确性，实施例5中同时对所得序列进行了测序，结果如表4所示，有效信号区域均与预期序列对比一致，无错配及缺口/插入子。
表3.制备组装体所用的RNA短链的序列信息
Figure PCTCN2021098422-appb-000001
表4.所制备267nt ssRNA的测序信息
测序方向 测得碱基数 对比碱基区域 错配数目 缺口/插入子数目
正向 241 40-264 0 0
反向 244 2-231 0 0
实施例6.含有精准定点修饰的267nt RNA单链的制备
为了合成含有精准定点修饰的RNA序列，实施例6中将长达267/220bp的RNA双链进行分割，短链分割方式基本同实施例5所述。其中，在R-n15、R-n16或R-n17三条短链中任取一条引入一个m 6A修饰(含有修饰的3条RNA序列分别称为R-n15-m 6A、R-n16-m 6A及R-n17-m 6A)，或在R-n15、R-n17、R-n20或R-n21四条短链中任取一条引入一个BrU修饰(含有修饰的4条RNA序列分别称为R-n15-BrU、R-n17-BrU、R-n20-BrU、R-n21-BrU)。简便起见，仅将所用修饰序列示于表5，其余未修饰序列依然与实施例5中所述相同。需要说明的是，本实验中所用R-n与R-x系列短链均带有5’末端磷酸基团修饰(R-n16及其对应的碱基修饰短链除外)，R-m与R-y系列短链均无修饰。
将实施例5中所述的18条短链等摩尔比混合并进行组装，其中非修饰短链R-n15、R-n16或R-n17分别被修饰短链R-n15-m 6A、R-n16-m 6A及R-n17-m 6A替代，非修饰短链R-n15、R-n17、R-n20或R-n21则分别被修饰短链R-n15-BrU、R-n17-BrU、R-n20-BrU、R-n21-BrU替代，每组仅替代一条序列，一共组装7组。组装体的合成条件与实施例1中相同。获得7组带有不同位点修饰的RNA267/220组装体水溶液后，通过T4 RNA ligase对链1中的9个连接口位点进行连接，使链1中的所有短链分别形成完整的、带有精准定点修饰的267nt链。所得连接完成的链1以聚丙烯酰胺凝胶电泳进行观察，结果如图9所示。
如图9所示，利用该方法，可以一锅内制备出在不同位置带有精准定点修饰的267nt RNA单链。
表5.制备含有精准定点修饰的267nt RNA所用的7条待修饰RNA短链的序列信息
序列名称 序列信息 nt SEQ ID NO
R-n15-m 6A AUUAAAGGUUUAUACCUUCCCA(m6A)GGU 25 SEQ ID NO：47
R-n16-m 6A AACAAA(m6A)CCAACCAACUUUCGAUCU 24 SEQ ID NO：48
R-n17-m 6A CUUGUA(m6A)GAUCUGUUCUCUAAACGA 24 SEQ ID NO：49
R-n15-BrU AUUAAAGGUUUAUACCUU(BrU)CCCAGGU 25 SEQ ID NO：50
R-n17-BrU CUU(BrU)GUAGAUCUGUUCUCUAAACGA 24 SEQ ID NO：51
R-n20-BrU UUACGGUU(BrU)UCGUCCGUGUUGCAGC 24 SEQ ID NO：52
R-n21-BrU CGAUCAUCAGCACAUCUAGGU(BrU)UUC 24 SEQ ID NO：53
本公开的上述实施例仅是为清楚地说明本公开所作的举例，而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本公开权利要求的保护范围之内。

Claims (16)

  1. 一种制备长链RNA的方法，其包括以下步骤：
    合成步骤：合成第一链的核酸片段组和第二链的核酸片段组，所述第一链的核酸片段组由RNA片段组成，所述第二链的核酸片段组由RNA片段和DNA片段中的至少一种组成；
    所述第一链的核酸片段组包括RNA片段组a和任选的RNA片段组b，所述第二链的核酸片段组包括核酸片段组c和任选的核酸片段组d；所述RNA片段组a包括RNA片段n i和RNA片段n i+1，所述核酸片段组b包括RNA片段x ii和RNA片段x ii+1，所述核酸片段组c包括核酸片段m i，所述核酸片段组d包括核酸片段y ii和核酸片段y ii+1中的至少一种，i、ii彼此独立的选自1以上的整数；
    其中，核酸片段m i的5’端序列与RNA片段n i+1的5’端序列为互补序列，核酸片段m i的3’端序列与RNA片段n i的3’端序列为互补序列；RNA片段x ii和RNA片段x ii+1的部分碱基互补形成具有5’延伸臂和3’延伸臂的发夹结构，所述5’延伸臂序列与核酸片段y ii的5’端序列为互补序列，所述3’延伸臂序列与核酸片段y ii+1的3’端序列为互补序列；
    退火步骤：将所述第一链的核酸片段组和第二链的核酸片段组混合于同一反应体系中，退火，形成双链组装体前体；其中，所述第一链中相邻的两个核酸片段之间存在连接口，所述第二链中相邻的两个核酸片段之间存在连接口；所述第一链的核酸片段组中的相邻核酸片段之间的连接口与所述第二链的核酸片段组中的相邻核酸片段之间的连接口相互错开；
    连接步骤：连接所述第一链的连接口，得到由连续化的单链RNA与片段化的单链核酸链互补形成的双链组装体。
  2. 根据权利要求1所述的制备长链RNA的方法，其中，所述方法还包括如下步骤：
    变性步骤：对所述双链组装体进行变性处理，得到连续化的单链RNA；
    可选地，所述方法还包括纯化步骤：从所述反应体系中纯化所述连续化的单链RNA。
  3. 根据权利要求1或2所述的制备长链RNA的方法，其中，所述发夹结构还包括形成双链结构的茎干区和未形成双链结构的茎环区，所述茎干区的5’末端和3’末端分别连接所述5’延伸臂和所述3’延伸臂；优选的，所述RNA片段x ii和RNA片段x ii+1之间的连接口位于所述茎干区。
  4. 根据权利要求1-3任一项所述的制备长链RNA的方法，其中，
    所述RNA片段n i+1的3’端序列与所述核酸片段组c的其它核酸片段的3’端序列为互补序列或为未配对序列；或者，
    所述核酸片段y ii的3’端序列与所述RNA片段组a的其它核酸片段的3’端序列为互补序列或为未配对序列；或者，
    所述核酸片段y ii+1的5’端序列与所述RNA片段组a的其它核酸片段的5’端序列为互补序列或为未配对序列；
    可选地，所述RNA片段n i+1的3’端序列与核酸片段m i+1的3’端序列为互补序列，所述核酸片段m i+1的5’端序列与所述核酸片段组a的其它核酸片段为互补序列或为未配对序列。
  5. 根据权利要求1-4任一项所述的制备长链RNA的方法，其中，所述连续化的单链RNA的长度为60nt以上，优选60-1000nt。
  6. 根据权利要求1-5任一项所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的长度为8-120nt，优选10-80nt，更优选15-40nt，最优选20-30nt。
  7. 根据权利要求1-6任一项所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的5’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt；或者，
    所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的3’端序列的长度为4nt以上，优选4-50nt，更优选6-30nt，最优选10-20nt。
  8. 根据权利要求1-7任一项所述的制备长链RNA的方法，其中，所述第一链的核酸片段组中任一核酸片段包含位于5’末端的磷酸基团，和位于3’末端的羟基；所述连接步骤中，将所述连接口两侧的磷酸基团和羟基连接为磷酸二酯键；
    可选地，以酶连接或化学连接将所述相邻的磷酸基团和羟基连接为磷酸二酯键。
  9. 根据权利要求1-8任一项所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的碱基，且紧邻所述连接口的位置处的碱基为未修饰的碱基；
    可选地，所述修饰选自m 6A、Ψ、m 1A、m 5A、ms 2i 6A、i 6A、m 3C、m 5C、ac 4C、m 7G、m2,2G、m 2G、m 1G、Q、m 5U、mcm 5U、ncm 5U、ncm 5Um、D、mcm 5s 2U、Inosine(I)、hm 5C、s 4U、s 2U、偶氮苯、Cm、Um、Gm、t 6A、yW、ms 2t 6A或其衍生物。
  10. 根据权利要求1-9任一项所述的制备长链RNA的方法，其中，所述第一链的核酸片段组和所述第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的核糖，且紧邻所述连接口的位置处的核糖为未修饰的核糖；
    可选地，所述修饰选自LNA、2’-OMe、3’-OMeU、vmoe、2'-F或2’-OBn(2’-O-benzyl group)或其衍生物。
  11. 根据权利要求1-10任一项所述的制备长链RNA的方法，其中，所述第一链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的磷酸二酯键，且紧邻所述连接口的位置处的磷酸二酯键为未修饰的磷酸二酯键；
    或者，所述第二链的核酸片段组中任一核酸片段的一个或多个位置处包含修饰的磷酸二酯键，且紧邻所述连接口的位置处的磷酸二酯键为未修饰的磷酸二酯键；
    可选地，所述修饰选自phosphorothioate(PS)、nucleotide triphosphate(NTPαS)或其衍生物。
  12. 根据权利要求1-11任一项所述的制备长链RNA的方法，其中，所述退火步骤中，将所述第一链的核酸片段组和所述第二链的核酸片段组孵育后，降温，形成双链组装体前体；
    可选地，所述孵育的温度为0-100℃的任意温度，优选10-85℃的任意温度，更优选20-65℃区间内的任意温度。
  13. 根据权利要求1-12任一项所述的制备长链RNA的方法，其中，所述退火步骤中，将所述第一链的核酸片段组和第二链的核酸片段组溶解于同一溶剂中，得到所述反应体系。
  14. 根据权利要求13所述的制备长链RNA的方法，其中，所述反应体系的pH为3-11，优选pH 4-10，更优选pH 5-9，最优选pH 6-8。
  15. 根据权利要求13或14所述的制备长链RNA的方法，其中，所述反应体系中，所述第一链的核酸片段组和第二链的核酸片段组中任意两个核酸片段的摩尔比为1：(0.1-10)，优选1：(0.5-2)，最优选1：1。
  16. 一种长链RNA，其中，所述长链RNA由权利要求1-15任一项所述的方法制得，所述长链RNA为单链的长链RNA；
    优选地，所述长链RNA的一个或多个位置处包含修饰的碱基、核糖或磷酸二酯键。
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