Efficient RNA enzymatic ligation method

Abstract

An efficient RNA enzymatic ligation method. Specifically, provided is a method for preparing a target single-stranded RNA, the method comprising: providing a plurality of RNA fragments on the basis of a target single-stranded RNA; providing a plurality of DNA fragments on the basis of the plurality of RNA fragments to form a plurality of RNA / DNA hybrid double-stranded fragments, wherein the DNA fragments and the corresponding RNA fragments form RNA / DNA hybrid double-stranded fragments, the RNA / DNA hybrid double-stranded fragments contain base-complementary regions, and one end or both ends of the RNA / DNA hybrid double-stranded fragments have overhangs; ligating the plurality of RNA / DNA hybrid double-stranded fragments in the presence of a ligase polypeptide to form a longer RNA / DNA hybrid double strand with the aid of the base complementary pairing of the overhangs, wherein the RNA / DNA hybrid double strand comprises the target single-stranded RNA; and removing the DNA strand or the DNA fragments from the RNA / DNA hybrid double strand to obtain the target single-stranded RNA.

Description

An efficient RNA ligation method TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an efficient RNA ligation method. BACKGROUND

[0002] At present, the commonly used method for the synthesis of long-chain RNA is in vitro transcription and ligase synthesis. In vitro transcription is the mainstream preparation method in the mRNA vaccine and drug industry, but its synthesis cycle is long, and it cannot perform site-specific modifications such as methylation and thiation in the sequence, and can only incorporate base analogs during transcription to form an mRNA sequence containing modifications, but the incorporation site cannot be selected, which restricts RNA drug research and development.

[0003] Ligase synthesis is a method of assembling chemically synthesized oligonucleotide fragments and using the characteristics of ligase to connect short fragments into long fragments. However, due to the particularity of RNA ligase, there are limitations in connection efficiency, especially when dealing with longer RNA molecules, the secondary structure of RNA (such as hairpin structure) may affect the accessibility of the enzyme and the availability of the substrate, thereby reducing the connection efficiency. In addition, although RNA ligase has a certain sequence specificity, it may be difficult to ensure only the connection of target sequences in a complex RNA mixture, and non-specific connection may occur. The existing method does not have universality and process amplification characteristics.

[0004] In summary, long-chain RNA with modifications is widely needed in the fields of gene expression regulation and drug development, and traditional chemical synthesis and in vitro transcription methods cannot meet market demand. The conventional enzymatic ligation method is limited by the secondary structure of RNA sequence and sequence base preference and cannot be applied to the ligation of various sequences. Therefore, it is necessary to develop a connection method that is not affected by sequence secondary structure and base difference, which is more conducive to large-scale preparation and industrial conversion. SUMMARY

[0005] The present application provides a long-chain RNA ligation method, which can significantly improve the ligation efficiency of RNA ligase, the purification efficiency of single-stranded RNA, or reduce sequence preference.

[0006] The long-chain RNA ligation method comprises the following steps: designing a plurality of RNA fragments according to a target single-stranded RNA, and designing DNA fragments that can pair with the RNA fragments; forming RNA / DNA hybrid double-stranded fragments by base complementary pairing between the DNA fragments and the corresponding RNA fragments; connecting the plurality of RNA / DNA hybrid double-stranded fragments to form a complete long-chain RNA / DNA hybrid double strand; and removing the DNA strand or DNA fragments in the double strand to obtain the target single-stranded RNA.

[0007] The method is named zigzag ligation method, and each RNA / DNA hybrid double-stranded fragment can be called a zigzag structure. The zigzag ligation method can completely open the RNA secondary structure by forming RNA / DNA double-stranded fragments, thereby significantly improving the ligation efficiency of the RNA ligase. The zigzag ligation method can also be paired and ligated by sticky ends, and a stable double-stranded structure can be formed after ligation. In addition, the zigzag ligation method can have less sequence bias.

[0008] On the other hand, the structure can significantly improve the HPLC purification efficiency when performing HPLC purification, because it is not interfered by complex secondary structure sequences. After purification, the single-stranded DNA is digested, and the final single-stranded RNA product is obtained.

[0009] In one aspect, the application provides a method for preparing a target single-stranded RNA, comprising:

[0010] (1) providing a plurality of RNA fragments according to the target single-stranded RNA;

[0011] (2) providing a plurality of DNA fragments according to the plurality of RNA fragments, to form a plurality of RNA / DNA hybrid double-stranded fragments, wherein the DNA fragments and the corresponding RNA fragments form the RNA / DNA hybrid double-stranded fragments, wherein the RNA / DNA hybrid double-stranded fragments have a base complementary region, and one end or both ends of the RNA / DNA hybrid double-stranded fragments have overhanging structures;

[0012] (3) ligating the plurality of RNA / DNA hybrid double-stranded fragments in the presence of a ligase polypeptide to form a complete long-chain RNA / DNA hybrid double-stranded, wherein the RNA / DNA hybrid double-stranded includes the target single-stranded RNA;

[0013] (4) removing the DNA strand or DNA fragment in the RNA / DNA hybrid double-stranded to obtain the target single-stranded RNA.

[0014] In some embodiments, the length of the RNA fragment is 20-10000 nt.

[0015] In some embodiments, the length of the overhanging structure is 4-20 nt.

[0016] In some embodiments, the plurality of RNA / DNA hybrid double-stranded fragments includes an RNA / DNA hybrid double-stranded fragment 1 and an RNA / DNA hybrid double-stranded fragment 2 adjacent thereto, wherein the 3' end of the RNA fragment of the RNA / DNA hybrid double-stranded fragment 1 and the 5' end of the RNA fragment of the RNA / DNA hybrid double-stranded fragment 2 are connected to form a continuous part of the target single-stranded RNA.

[0017] In some embodiments, the overhang structure at the 5' end of the DNA segment of the RNA / DNA hybrid double-stranded segment 1 is base complementary to the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded segment 2.

[0018] In some embodiments, the method of the single-stranded RNA, wherein

[0019] a) the overhang structure at the 5' end of the DNA segment of the RNA / DNA hybrid double-stranded segment 1 is base complementary to the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded segment 2; or

[0020] b) the overhang structure at the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded segment 1 is base complementary to the overhang structure at the 3' end of the DNA segment of the RNA / DNA hybrid double-stranded segment 2.

[0021] In some embodiments, the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded segment 1 has a hydroxyl modification.

[0022] In some embodiments, the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded segment 2 has a phosphate modification.

[0023] In some embodiments, the DNA segment has no modification or a hydroxyl modification at both ends.

[0024] In some embodiments, the 5' end and / or 3' end of the DNA includes an amino modification.

[0025] In some embodiments, the plurality of RNA / DNA hybrid double-stranded segments includes an RNA / DNA hybrid double-stranded 5' end segment and / or an RNA / DNA hybrid double-stranded 3' end segment, wherein the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded 5' end segment is a blunt end and the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded 3' end segment is a blunt end.

[0026] In some embodiments, the ligase polypeptide includes an RNA ligase polypeptide or a DNA ligase polypeptide.

[0027] In some embodiments, the RNA ligase polypeptide is not limited to T4 RNA ligase 2. The ligase polypeptide can be from eukaryotes, prokaryotes and / or bacteriophages, including but not limited to one or more of the following groups: T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase, eukaryotic DNA ligase I, eukaryotic DNA ligase III, eukaryotic DNA ligase IV, E. coli DNA ligase, Hi-T4 TM DNA ligase, Salt-T4 DNA ligase, 9°N TM DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ligase, thermostable 5' App DNA / RNA Ligase, RtcB ligase and PBCV DNA ligase. The ligase polypeptide also includes isozymes of any ligase in the above groups and / or fragments of any ligase that can perform ligation function.

[0028] In some embodiments, step (4) further comprises: removing the DNA strand or DNA fragment in the RNA / DNA hybrid duplex in the presence of a DNA enzyme, to obtain the target single-stranded RNA.

[0029] In some embodiments, further comprising: providing N RNA fragments according to the target single-stranded RNA, wherein the N RNA fragments are connected to form the complete target single-stranded RNA.

[0030] In some embodiments, N is an integer greater than or equal to 2.

[0031] In some embodiments, further comprising, providing N DNA fragments, wherein the N DNA fragments correspond one-to-one to the N RNA fragments to form N RNA / DNA hybrid duplex fragments.

[0032] In some embodiments, the CG content of the RNA fragments is between 0-100%.

[0033] In some embodiments, the overhang structure is a free linear structure without any secondary structure.

[0034] In some embodiments, the temperature range for forming the plurality of RNA / DNA hybrid duplex fragments is 4°C-37°C.

[0035] In some embodiments, the temperature range for connecting the plurality of RNA / DNA hybrid duplex fragments to form the RNA / DNA hybrid duplex is 4°C-37°C.

[0036] In some embodiments, wherein the forming RNA / DNA hybrid duplexes or forming RNA / DNA hybrid duplex fragments further comprises: purifying by reverse phase HPLC purification or ion HPLC purification to obtain the successfully ligated RNA / DNA hybrid duplexes.

[0037] In some embodiments, wherein the reverse phase HPLC purification comprises, the polarity of the stationary phase of the reverse phase HPLC purification is less than the polarity of the mobile phase.

[0038] In some embodiments, wherein the stationary phase of the reverse phase HPLC purification is C8 or C18.

[0039] In some embodiments, wherein the mobile phase is a combination of reverse phase buffer A and reverse phase buffer B, wherein the reverse phase buffer A is a solution of triethylammonium carbonate (TEAB), triethylamine (TEA) or triethylammonium acetate (TEAA).

[0040] In some embodiments, wherein the concentration of triethylammonium carbonate (TEAB), triethylamine (TEA) or triethylammonium acetate (TEAA) ranges from 50-150 mM.

[0041] In some embodiments, wherein the reverse phase buffer B is an acetonitrile solution of buffer A.

[0042] In some embodiments, wherein the flow rate of the mobile phase is 0.8-1.2 ml / min.

[0043] In some embodiments, wherein the volume ratio of the reverse phase buffer B in the mobile phase is 10%-40% at 43 minutes before the gradient elution.

[0044] In some embodiments, wherein the ion HPLC purification comprises, the stationary phase of the ion HPLC purification is an ion exchange medium.

[0045] In some embodiments, wherein the stationary phase of the ion HPLC purification is an anion exchange medium.

[0046] In some embodiments, wherein the stationary phase of the ion HPLC purification is a weak anion exchange medium DEAE.

[0047] In some embodiments, wherein the stationary phase of the ion HPLC purification is a strong anion exchange medium Q.

[0048] In some embodiments, wherein the mobile phase is a combination of ion buffer A and ion buffer B, wherein the ion buffer A comprises urea and phosphate buffer, or urea and TRIS buffer.

[0049] In some embodiments, wherein the ion buffer A comprises urea and phosphate buffer.

[0050] In some embodiments, the ion buffer A comprises urea and TRIS buffer.

[0051] In some embodiments, the concentration of the urea is 0-8 M.

[0052] In some embodiments, the concentration of the phosphate buffer and the TRIS buffer is 50-150 mM.

[0053] In some embodiments, the ion buffer B comprises urea, phosphate buffer or TRIS, guanidine hydrochloride or NaCl solution.

[0054] In some embodiments, the ion buffer B comprises urea, phosphate buffer and guanidine hydrochloride solution.

[0055] In some embodiments, the ion buffer B comprises urea, phosphate buffer and NaCl solution.

[0056] In some embodiments, the ion buffer B comprises urea, TRIS and guanidine hydrochloride solution.

[0057] In some embodiments, the ion buffer B comprises urea, TRIS and NaCl solution.

[0058] In some embodiments, the flow rate of the mobile phase is 0.8-1.2 ml / min.

[0059] In some embodiments, the volume ratio of the reverse phase buffer B in the mobile phase is 10%-80% at 63 minutes before the gradient elution.

[0060] In some embodiments, the purification temperature is 25-60 °C.

[0061] In another aspect, the present application provides the target single-stranded RNA obtained by the above method.

[0062] In another aspect, the present application provides the use of the above method for obtaining the target single-stranded RNA.

[0063] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description. In some instances, detailed descriptions of well-known methods and apparatuses are omitted so as not to obscure the description of the present application with unnecessary detail. Nothing herein is to be construed as an admission that the application is not entitled to antedate such disclosure by virtue of prior application. BRIEF DESCRIPTION OF DRAWINGS

[0064] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the detailed description of exemplary embodiments and the attached drawings. The drawings are briefly described as follows:

[0065] Figure 1 shows a schematic diagram of zigzag single connection described in the present application;

[0066] Figure 2 shows a schematic diagram of zigzag multi-batch connection for long chain splicing described in the present application;

[0067] Figure 3 shows a secondary structure prediction diagram of sgRNA described in the present application;

[0068] Figure 4 shows the detection results of urea-PAGE of traditional enzymatic ligation and zigzag ligation described in the present application;

[0069] Figure 5 shows the detection results of urea-PAGE of different modified DNA for zigzag ligation described in the present application;

[0070] Figure 6 shows the detection results of urea-PAGE of different length interfaces described in the present application;

[0071] Figure 7 shows the detection results of urea-PAGE of different reaction temperatures described in the present application;

[0072] Figure 8 shows the detection results of urea-PAGE of different concentrations of different ligases described in the present application;

[0073] Figure 9 shows the detection results of urea-PAGE of different modified oligonucleotide chains described in the present application;

[0074] Figure 10 shows the HPLC spectrum of reverse phase purification described in the present application;

[0075] Figure 11 shows the urea-PAGE spectrum of reverse phase purification described in the present application;

[0076] Figure 12 shows the HPLC spectrum of ion purification described in the present application;

[0077] Figure 13 shows the urea-PAGE spectrum of ion purification described in the present application;

[0078] Figures 14-15 show the linking results of different ligases described in the present application. DETAILED DESCRIPTION

[0079] The embodiments of the invention of the present application are illustrated by the following specific examples, and other advantages and effects of the invention of the present application can be easily understood by those skilled in the art from the disclosure of the specification.

[0080] Terminology Definition

[0081] The term "single strand" as used in this application refers to a single polymeric nucleic acid chain formed by the linkage of multiple nucleotide monomers, which can be linked by phosphodiester bonds between nucleotides. The single strand can be formed by the condensation linkage of a phosphate group at the 5' end and a hydroxyl group at the 3' end of a nucleotide monomer. The nucleotide monomers can be ribonucleotide monomers, and the resulting single strand can be an RNA single strand; or the nucleotide monomers can be deoxyribonucleotide monomers, and the resulting single strand can be a DNA single strand.

[0082] The "hybrid double strand" described in this application refers to a double-stranded nucleic acid molecule composed of two anticomplementary polynucleotide single strands, which can be linked together by hydrogen bonds formed between one or more complementary bases on the double strands. Depending on the type of single strand, it can be classified as DNA-DNA double strand (containing two DNA single strands) or RNA-DNA double strand (containing one DNA single strand and one RNA single strand). In some embodiments, the two anticomplementary polynucleotide single strands can be perfectly complementary, i.e., there are no unpaired bases at either end of the single strand. In some embodiments, the two anticomplementary polynucleotide single strands can be imperfectly complementary, i.e., there are one or more unpaired bases at either end or one end of either single strand.

[0083] The "ligase polypeptide" described in this application refers to an enzyme or polypeptide capable of catalyzing the formation of phosphodiester bonds between nucleotides, thereby linking nucleic acid fragments together, between nucleic acid fragments and free nucleotides, or between bases of free nucleotides. The ligase polypeptide catalyzes the ligation reaction on single-stranded or hybridized double-stranded nucleic acids, and can catalyze the formation of phosphodiester bonds at the sticky or blunt ends of the nucleic acid strand. The ligase polypeptide can be a ligase, such as DNA ligase or RNA ligase, or a functional fragment of the aforementioned ligases, wherein the functional fragment can be any fragment of the aforementioned ligases capable of performing the ligation function. The ligase polypeptide can be ATP-dependent and / or NAD+-dependent. + Dependent on specific types. The ligase polypeptide may be derived from eukaryotes, prokaryotes, and / or bacteriophages, including but not limited to one or more of the following groups: T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase, eukaryotic DNA ligase I, eukaryotic DNA ligase III, eukaryotic DNA ligase IV, E. coli DNA ligase, Hi-T4. TM DNA ligase, Salt-T4 DNA ligase, 9°N TM DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase ligase, a thermostable 5' App DNA / RNA ligase, a RtcB ligase, and a PBCV DNA ligase. The ligase polypeptide also includes an isozyme of any of the ligases described above and / or a fragment of any of the ligases that is capable of performing ligation.

[0084] The "DNAase" as used herein refers to an endonuclease that can cleave the phosphodiester bond between deoxynucleotides of a single strand or a double strand of DNA. Thus, the cleavage produces free monodeoxynucleotides or oligodeoxynucleotides of single strand or double strand. The product of the cleavage of a single strand or a double strand of DNA by the DNAase has a phosphate group at the 5' end and a hydroxyl group at the 3' end. The DNA strand cleaved by the DNAase can be a DNA-DNA hybridized double strand or a DNA-RNA hybridized double strand. When the DNAase cleaves a DNA-RNA hybridized double strand, it produces a RNA single strand without cleavage. The DNAase can be from a eukaryote, a prokaryote, and / or a bacteriophage.

[0085] The "overhang structure" or "sticky end" or "overhang" as used herein refers to the end of one side or both sides of a hybridized double strand of nucleic acid, in which one strand has one or more unpaired nucleotides overhanging. The overhang structure can be at any end of either strand of a double strand of nucleic acid. For example, when the hybridized double strand is a DNA-RNA hybridized double strand, the overhang structure can be at the end of one side of the RNA single strand. In some embodiments, when the hybridized double strand is a DNA-RNA hybridized double strand, only the end of one side of the RNA single strand contains the overhang structure and the other side is a blunt end. In some embodiments, when the hybridized double strand is a DNA-RNA hybridized double strand, the ends of both sides of the hybridized double strand contain the overhang structure. The overhang structure can include a single unpaired nucleotide or multiple unpaired nucleotides, such as 6-20 unpaired nucleotides. In some embodiments, the overhang structure contains 6-16 unpaired nucleotides. The overhang structure is a linear structure without any secondary structure.

[0086] The "blunt end" as used herein generally refers to an end of a hybridized double strand with completely complementary bases, i.e., there is no unpaired base at the blunt end of the hybridized double strand. The hybridized double strand can have a blunt end on one side or on both sides. The blunt end can be ligated under the catalysis of a ligase, such as T4 DNA ligase. The ligation efficiency of the blunt end is much lower than that of the sticky end, and the zigzag structure will seek a sticky end to pair and ligate. Moreover, the blunt end does not contain a 5' phosphate group modification, and there is no blunt end ligation.

[0087] The "weak denaturing reverse phase HPLC purification method" refers to a HPLC purification method for separating oligonucleotide chains of different lengths (with or without modification groups) according to the hydrophobicity of the separated substances. Reverse phase HPLC is a very effective purification method for purifying target sequences with hydrophobic modification groups and non-target sequences without hydrophobic modification groups. For zigzag structures, the weak denaturing reverse phase purification method can maintain the zigzag RNA-DNA hybrid double-stranded structure, effectively remove short chains that are not properly connected, and only purify the zigzag RNA-DNA hybrid double-stranded structure that is successfully connected together.

[0088] The "ion exchange HPLC purification method" refers to a HPLC purification method for separating target sequences from non-target sequences according to the amount of negative charge on the molecules using gradient elution. Different lengths of zigzag structures can be distinguished by ion exchange HPLC purification method, and the zigzag RNA-DNA hybrid double-stranded structure will not be opened, thereby distinguishing between successfully connected and unsuccessfully connected fragments.

[0089] In this application, the term "about" means approximately, in the vicinity of, roughly, or around. When the term "about" is used in conjunction with a numerical value or a range of numerical values, it can be modified and expanded by extending the upper and lower limits of the listed numerical value or range of numerical values. Generally, the term "about" is used herein to modify a numerical value by a variation of 10%, 8%, 5%, 2%, 1%, 0.5%, or 0.1% above and below the specified value.

[0090] DETAILED DESCRIPTION

[0091] The application provides a long-chain RNA ligation method that can significantly improve the ligation efficiency of RNA ligase, the purification efficiency of single-stranded RNA, or reduce sequence bias.

[0092] The long-chain RNA ligation method designs multiple RNA fragments according to the target single-stranded RNA, and designs DNA fragments that can pair with the RNA fragments. Through base complementary pairing, the DNA fragments and the corresponding RNA fragments form RNA / DNA hybrid double-stranded fragments. Then, multiple RNA / DNA hybrid double-stranded fragments are connected to form a complete long-chain RNA / DNA hybrid double-stranded structure. Finally, the DNA strand or DNA fragment in the double-stranded structure is removed to obtain the target single-stranded RNA. The ligation strategy is shown in FIG. 1 and / or FIG. 2.

[0093] For the synthesis of long single-stranded RNA molecules, the prior art has attempted to use one long DNA template strand and multiple RNA fragments to form a long single-stranded RNA with gaps by base pairing, and then use RNA ligase to connect the gaps with phosphodiester bonds. Although this method can obtain single-stranded RNA, it does not essentially solve the problem of RNA secondary structure.

[0094] The method used in the present application eliminates the RNA end secondary structure (for example, the end secondary structure shown in Figure 3) by forming multiple RNA / DNA hybrid double-stranded fragments and then connecting the multiple RNA / DNA hybrid double-stranded fragments to form a complete long-chain RNA / DNA hybrid double strand, significantly improving the connection efficiency of RNA ligase.

[0095] In addition, the purification efficiency of double-stranded RNA / DNA is higher and the method is simpler.

[0096] For example, as shown in Figure 1, in order to obtain the target single-stranded RNA (i.e., "single-stranded RNA ligation product"), two single-stranded RNA fragments (i.e., first single-stranded RNA and second single-stranded RNA) can be designed and provided according to the target single-stranded RNA. In order to avoid the secondary structure of the single-stranded RNA fragments, DNA fragments (i.e., first single-stranded DNA and second single-stranded DNA) that pair with the first single-stranded RNA and the second single-stranded RNA, respectively, are used to form first RNA / DNA hybrid double-stranded fragments and second RNA / DNA hybrid double-stranded fragments, respectively. Then the first RNA / DNA hybrid double-stranded fragments and the second RNA / DNA hybrid double-stranded fragments are connected under the action of T4 RNA ligase 2 or other ligases to form an RNA / DNA hybrid double strand, and at this time the RNA / DNA hybrid double strand includes a complete ligation target single-stranded RNA (i.e., "single-stranded RNA ligation product")

[0097] If direct ligation with ligase is used, when the single-stranded RNA fragment is too long (for example, greater than 20 nt), secondary structures will be generated at both ends of the single-stranded RNA (for example, as shown in Figure 3), and the secondary structure of the RNA (such as a hairpin structure) can affect the accessibility of the enzyme and the availability of the substrate, thereby reducing the ligation efficiency. In addition, the long-chain RNA formed under the condition of low yield will also become difficult to purify due to the presence of secondary structures (whether the secondary structure of the fragment RNA or the secondary structure of the formed long-chain RNA).

[0098] In one aspect, the present application provides a method for preparing a target single-stranded RNA, comprising: providing a plurality of RNA fragments according to the target single-stranded RNA; providing a plurality of DNA fragments according to the plurality of RNA fragments, forming a plurality of RNA / DNA hybrid double-stranded fragments, wherein the DNA fragments and the corresponding RNA fragments form the RNA / DNA hybrid double-stranded fragments, wherein the RNA / DNA hybrid double-stranded fragments have a base complementary region, and one end or both ends of the RNA / DNA hybrid double-stranded fragments have overhang structures; connecting the plurality of RNA / DNA hybrid double-stranded fragments in the presence of a ligase polypeptide to form an RNA / DNA hybrid double strand, wherein the RNA / DNA hybrid double strand comprises the target single-stranded RNA; removing the DNA strand or DNA fragment in the RNA / DNA hybrid double strand to obtain the target single-stranded RNA.

[0099] Fragment

[0100] The single-stranded RNA preparation method described in the present application, wherein the fragments include RNA fragments designed according to the target single-stranded RNA, corresponding DNA fragments hybridized with the RNA fragments, and RNA / DNA hybrid double-stranded fragments.

[0101] Wherein, the RNA fragments designed according to the target single-stranded RNA can be N kinds, and the connection of the N kinds of RNA fragments can form the complete target single-stranded RNA. Wherein N can be any integer greater than or equal to 2, for example, N can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, that is, the RNA fragments designed according to the target single-stranded RNA can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 kinds.

[0102] Correspondingly, the corresponding DNA fragments hybridized with the RNA fragments and the RNA / DNA hybrid double-stranded fragments have the same number of types as the RNA fragments, and can also be N kinds. Wherein the N kinds of DNA fragments can correspond to the N kinds of RNA fragments one by one, and can form N kinds of RNA / DNA hybrid double-stranded fragments. Wherein N can be any integer greater than or equal to 2, for example, N can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, that is, the corresponding DNA fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 kinds, and the RNA / DNA hybrid double-stranded fragments can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 kinds.

[0103] The ends of the RNA fragments, DNA fragments and / or RNA / DNA hybrid double-stranded fragments can have a modification group. The modification group can include a hydroxyl group, a phosphate group, an amino group and any other modification group. The phosphate group modification can be a phosphate modification or a phosphorothioate modification.

[0104] The 3' end of the RNA fragment can have a hydroxyl group modification or a phosphate group modification.

[0105] The 5' end of the RNA fragment can have a hydroxyl group modification or a phosphate group modification.

[0106] In some embodiments, both ends of the DNA fragment have no modification or a hydroxyl group modification.

[0107] In some embodiments, the 5' end and / or the 3' end of the DNA includes an amino group modification.

[0108] For two adjacent RNA fragments, i.e. the 3' end of the first RNA fragment and the 5' end of the second RNA fragment can be connected to form a continuous part of the target single-stranded RNA, the 3' end of the first RNA fragment is a phosphate group modification and the 5' end of the second RNA fragment is a hydroxyl group modification. For two adjacent RNA fragments, the 3' end of the first RNA fragment is a hydroxyl group modification and the 5' end of the second RNA fragment is a phosphate group modification.

[0109] For two adjacent RNA / DNA hybrid double-stranded fragments, i.e. the RNA / DNA hybrid double-stranded fragment 1 and the RNA / DNA hybrid double-stranded fragment 2 adjacent thereto (wherein the 3' end of the RNA fragment of the RNA / DNA hybrid double-stranded fragment 1 and the 5' end of the RNA fragment of the RNA / DNA hybrid double-stranded fragment 2 are connected to form a continuous part of the target single-stranded RNA), the modification rules are as follows:

[0110] The 3' end of the RNA fragment of the hybrid double-stranded fragment 1 is a phosphate group modification, and the 5' end of the RNA fragment of the hybrid double-stranded fragment 2 is a hydroxyl group modification; or,

[0111] The 3' end of the RNA fragment of the hybrid double-stranded fragment 1 is a hydroxyl group modification, and the 5' end of the RNA fragment of the hybrid double-stranded fragment 2 is a phosphate group modification.

[0112] The purpose is to ensure that the RNA in the two adjacent RNA / DNA hybrid double-stranded fragments can be combined under the action of a ligase.

[0113] The plurality of RNA / DNA hybrid double-stranded fragments can comprise one RNA / DNA hybrid double-stranded 5' end fragment and / or one RNA / DNA hybrid double-stranded 3' end fragment. The plurality of RNA / DNA hybrid double-stranded fragments comprises one RNA / DNA hybrid double-stranded 5' end fragment and / or one RNA / DNA hybrid double-stranded 3' end fragment, wherein the 5' end of the RNA fragment of the RNA / DNA hybrid double-stranded 5' end fragment is a blunt end, and the 3' end of the RNA fragment of the RNA / DNA hybrid double-stranded 3' end fragment is a blunt end.

[0114] That is, for the RNA / DNA hybrid double-stranded fragment, with reference to the 3' end and 5' end of the RNA, the RNA / DNA hybrid double-stranded fragment can have a blunt end at the 3' end of the RNA and a sticky end at the 5' end of the RNA (which can occur when the RNA is the 3' end RNA of the target single-stranded RNA); the RNA / DNA hybrid double-stranded fragment can have a blunt end at the 5' end of the RNA and a sticky end at the 3' end of the RNA (which can occur when the RNA is the 5' end RNA of the target single-stranded RNA); the RNA / DNA hybrid double-stranded fragment can have a sticky end at the 3' end of the RNA and a sticky end at the 5' end of the RNA (which can occur when the RNA is the 5' end RNA of the target single-stranded RNA). As shown in FIG. 1 and FIG. 2.

[0115] wherein the length of the RNA fragment can be 20-1000 nt.

[0116] wherein the length of the RNA fragment can be 20-1500 nt. wherein the length of the RNA fragment can be 20-1000 nt. wherein the length of the RNA fragment can be 20-500 nt.

[0117] wherein the length of the RNA fragment can be 20-100, 20-99, 20-98, 20-97, 20-96, 20-95, 2-94, 20-93, 20-92, 20-91, 20-90, 20-89, 20-88, 20-87, 20-86, 20-85, 2-84, 20-83, 20-82, 20-81, 20-80, 20-79, 20-78, 20-77, 20-76, 20-75, 2-74, 20-73, 20-72, 20-71, 20-70, 20-69, 20-68, 20-67, 20-66, 20-65, 2-64, 20-63, 20-62, 20-61, 20-60, 20-59, 20-58, 20-57, 20-56, 20-55, 2-54, 20-53, 20-52, 20-51, 20-50, 20-49, 20-48, 20-47, 20-46, 20-45, 2-44, 20-43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36, 20-35, 2-34, 20-33, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 2-24, 20-23, 20-22, or 20-21 nt.

[0118] The CG content of the RNA fragments can be between 0 and about 100%.The CG content of the RNA segment can be about 100 to about 1%, about 100 to about 2%, about 100 to about 3%, about 100 to about 4%, about 100 to about 5%, about 100 to about 6%, about 100 to about 7%, about 100 to about 8%, about 100 to about 9%, about 100 to about 10%, about 100 to about 11%, about 100 to about 12%, about 100 to about 13%, about 100 to about 14%, about 100 to about 15%, about 100 to about 16%, about 100 to about 17%, about 100 to about 18%, about 100 to about 19%, about 100 to about 20%, about 100 to about 21%, about 100 to about 22%, about 100 to about 23%, about 100 to about 24%, about 100 to about 25%, about 100 to about 26%, about 100 to about 27%, about 100 to about 28%, about 100 to about 29%, about 100 to about 30%, about 100 to about 31%, about 100 to about 32%, about 100 to about 33%, about 100 to about 34%, about 100 to about 35%, about 100 to about 36%, about 100 to about 37%, about 100 to about 38%, about 100 to about 39%, about 100 to about 40%, about 100 to about 41%, about 100 to about 42%, about 100 to about 43%, about 100 to about 44%, about 100 to about 45%, about 100 to about 46%, about 100 to about 47%, about 100 to about 48%, about 100 to about 49%, about 100 to about 50%, about 100 to about 51%, about 100 to about 52%, about 100 to about 53%, about 100 to about 54%, about 100 to about 55%, about 100 to about 56%, about 100 to about 57%, about 100 to about 58%, about 100 to about 59%, about 100 to about 60%, about 100 to about 61%, about 100 to about 62%, about 100 to about 63%, about 100 to about 64%, about 100 to about 65%, about 100 to about 66%, about 100 to about 67%, about 100 to about 68%, about 100 to about 69%, about 100 to about 70%, about 100 to about 71%, about 100 to about 72%, about 100 to about 73%, about 100 to about 74%, about 100 to about 75%, about 100 to about 76%, about 100 to about 77%, about 100 to about 78%, about 100 to about 79%, about 100 to about 80%, about 100 to about 81%, about 100 to about 82%, about 100 to about 83%, about 100 to about 84%, about 100 to about 85%, about 100 to about 86%, about 100 to about 87%, about 100 to about 88%, about 100 to about 89%, about 100 to about 90%, about 100 to about 91%, about 100 to about 92%, about 100 to about 93%, about 100 to about 94%, about 100 to about 95%, about 100 to about 96%, about 100 to about 97%, about 100 to about 98%, or about 100 to about 99%.

[0119] The CG content of the RNA fragment can be between about 0% and about 100%. The CG content of the RNA fragment can be approximately 0% to about 1%, approximately 0% to about 2%, approximately 0% to about 3%, approximately 0% to about 4%, approximately 0% to about 5%, approximately 0% to about 6%, approximately 0% to about 7%, approximately 0% to about 8%, approximately 0% to about 9%, approximately 0% to about 10%, approximately 0% to about 11%, approximately 0% to about 12%, approximately 0% to about 13%, approximately 0% to about 14%, approximately 0% to about 15%, approximately 0% to about 16%, approximately 0% to about 17%, approximately 0% to about 18%, approximately 0% to about 19%, approximately 0% to about 20%, approximately 0% to about 21%, approximately 0% to about 22%, approximately 0% to about 23%, approximately 0% to about 24%, and so on. 0-approx. 25%, 0-approx. 26%, 0-approx. 27%, 0-approx. 28%, 0-approx. 29%, 0-approx. 30%, 0-approx. 31%, 0-approx. 32%, 0-approx. 33%, 0-approx. 34%, 0-approx. 35%, 0-approx. 36%, 0-approx. 37%, 0-approx. 38%, 0-approx. 39%, 0-approx. 40%, 0-approx. 41%, 0-approx. 42%, 0-approx. 43%, 0-approx. 44%, 0-approx. 45%, 0-approx. 46%, 0-approx. 47%, 0-approx. 48%, 0-approx. 49%, approximately 0-approx. 50%, 0-approx. 51%, 0-approx. 52%, 0-approx. 53%, 0-approx. 54%, 0-approx. 55%, 0-approx. 56%, 0-approx. 57%, 0-approx. 58%, 0-approx. 59%, 0-approx. 60%, 0-approx. 61%, 0-approx. 62%, 0-approx. 63%, 0-approx. 64%, 0-approx. 65%, 0-approx. 66%, 0-approx. 67%, 0-approx. 68%, 0-approx. 69%, 0-approx. 70%, 0-approx. 71%, 0-approx. 72%, 0-approx. 73%, 0-approx. 74%. Approximately 0-75%, approximately 0-76%, approximately 0-77%, approximately 0-78%, approximately 0-79%, approximately 0-80%, approximately 0-81%, approximately 0-82%, approximately 0-83%, approximately 0-84%, approximately 0-85%, approximately 0-86%, approximately 0-87%, approximately 0-88%, approximately 0-89%, approximately 0-90%, approximately 0-91%, approximately 0-92%, approximately 0-93%, approximately 0-94%, approximately 0-95%, approximately 0-96%, approximately 0-97%, approximately 0-98%, or approximately 0-99%.

[0120] The CG content of the RNA segment can be from about 0 to about 10%, from about 0 to about 20%, from about 0 to about 30%, from about 0 to about 40%, from about 0 to about 50%, from about 0 to about 60%, from about 0 to about 70%, from about 0 to about 80%, from about 0 to about 90%, from about 0 to about 100%, from about 10 to about 20%, from about 10 to about 30%, from about 10 to about 40%, from about 10 to about 50%, from about 10 to about 60%, from about 10 to about 70%, from about 10 to about 80%, from about 10 to about 90%, from about 10 to about 100%, from about 20 to about 30%, from about 20 to about 40%, from about 20 to about 50%, from about 20 to about 60%, from about 20 to about 70%, from about 20 to about 80%, from about 20 to about 90%, from about 20 to about 100%, from about 30 to about 40%, from about 30 to about 50%, from about 30 to about 60%, from about 30 to about 70%, from about 30 to about 80%, from about 30 to about 90%, from about 30 to about 100%, from about 40 to about 50%, from about 40 to about 60%, from about 40 to about 70%, from about 40 to about 80%, from about 40 to about 90%, from about 40 to about 100%, from about 50 to about 60%, from about 50 to about 70%, from about 50 to about 80%, from about 50 to about 90%, from about 50 to about 100%, from about 60 to about 70%, from about 60 to about 80%, from about 60 to about 90%, from about 60 to about 100%, from about 70 to about 80%, from about 70 to about 90%, from about 70 to about 100%, from about 80 to about 90%, from about 80 to about 100%, or from about 90 to about 100%. One end or both ends of the RNA / DNA hybrid duplex segment has an overhang. When both ends of the RNA / DNA hybrid duplex segment have an overhang, the lengths of the overhangs can be the same. When both ends of the RNA / DNA hybrid duplex segment have an overhang, the lengths of the overhangs can be different. When both ends of the RNA / DNA hybrid duplex segment have an overhang, the sequences of the two overhangs can be reverse complements.

[0121] The length of the overhang can be from 6 to 20 nt.

[0122] The length of the overhang structure can be 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, or 6-20 nt. The length of the overhang structure can be 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, or 7-20 nt. The length of the overhang structure can be 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, or 8-20 nt. The length of the overhang structure can be 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, or 9-20 nt. The length of the overhang structure can be 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, or 10-20 nt. The length of the overhang structure can be 11-12, 11-13, 11-14, 11-15, 11-16, 11-17, 11-18, 11-19, or 11-20 nt. The length of the overhang structure can be 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12-19, or 12-20 nt. The length of the overhang structure can be 13-14, 13-15, 13-16, 13-17, 13-18, 13-19, or 13-20 nt. The length of the overhang structure can be 14-15, 14-16, 14-17, 14-18, 14-19, or 14-20 nt. The length of the overhang structure can be 15-16, 15-17, 15-18, 15-19, or 15-20 nt. The length of the overhang structure can be 16-17, 16-18, 16-19, or 16-20 nt. The length of the overhang structure can be 17-18, 17-19, or 17-20 nt. The length of the overhang structure can be 18-19, or 18-20 nt. The length of the overhang structure can be 19-20 nt.

[0123] An overhang structure that is too long can cause secondary structure of the DNA / RNA hybrid duplex fragment. Therefore, the length of the overhang structure is at most 20 nt or at most 19 nt or at most 18 nt or at most 17 nt or at most 16 nt.

[0124] At the same time, an overhang structure that is too short can cause a decrease in ligation efficiency. Therefore, the length of the overhang structure is at least 2 nt or at least 3 nt or at least 4 nt.

[0125] The length of the overhang structure can be 4-16 nt.

[0126] The overhang structure is a free linear structure without strong secondary structure.

[0127] ligase polypeptide

[0128] The single-stranded RNA preparation method described in the present application comprises connecting the plurality of RNA / DNA hybrid double-stranded fragments by the ligase polypeptide to form RNA / DNA hybrid double-stranded.

[0129] The ligase polypeptide can be a ligase, such as a DNA ligase or an RNA ligase, or any functional fragment of any DNA ligase or RNA ligase, which can be any fragment of the above ligases capable of performing the ligation function.

[0130] The ligase polypeptide can be ATP-dependent and / or NAD + dependent.

[0131] The ligase polypeptide can be from eukaryotes, prokaryotes and / or bacteriophages. The ligase polypeptide can be from eukaryotes, the ligase polypeptide can be from prokaryotes, such as Escherichia coli, and the ligase polypeptide can be from bacteriophages.

[0132] The ligase polypeptide can include one or more of the following groups, but is not limited to: T4 DNA ligase, T4 RNA ligase (such as T4 RNA ligase 1 or T4 RNA ligase 2), T3 DNA ligase, T7 DNA ligase, eukaryotic DNA ligase I, eukaryotic DNA ligase III, eukaryotic DNA ligase IV, E. coli DNA ligase, Hi-T4 TM DNA ligase, Salt-T4 DNA ligase, 9°N TM DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ligase, thermostable 5' App DNA / RNA Ligase, RtcB ligase and PBCV DNA ligase. The ligase polypeptide also includes isoenzymes of any ligase in the above groups and / or fragments of any ligase capable of performing the ligation function.

[0133] ligation method

[0134] The preparation method of the target single-stranded RNA described in the present application comprises connecting the plurality of RNA / DNA hybrid double-stranded fragments in the presence of a ligase polypeptide to form RNA / DNA hybrid double-stranded, wherein the RNA / DNA hybrid double-stranded comprises a target single-stranded RNA.

[0135] The plurality of RNA / DNA hybrid double-stranded fragments comprises an RNA / DNA hybrid double-stranded fragment 1 and an RNA / DNA hybrid double-stranded fragment 2 adjacent to the RNA / DNA hybrid double-stranded fragment 1, wherein the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 1 and the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2 are connected to form a continuous part of the target single-stranded RNA. Wherein the overhang structure at the 5' end of the DNA segment of the RNA / DNA hybrid double-stranded fragment 1 is base complementary to the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2.

[0136] Wherein, the base complementary process can be:

[0137] a) the overhang structure at the 5' end of the DNA segment of the RNA / DNA hybrid double-stranded fragment 1 is base complementary to the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2; or

[0138] b) the overhang structure at the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 1 is base complementary to the overhang structure at the 3' end of the DNA segment of the RNA / DNA hybrid double-stranded fragment 2.

[0139] Wherein, the connection can further comprise a connection between nucleotides under the action of a ligase polypeptide.

[0140] The connection between nucleotides can be:

[0141] a) the phosphate group of the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2 is connected to the hydroxyl group of the overhang structure at the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 1; and / or

[0142] b) the phosphate group of the overhang structure at the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 1 is connected to the hydroxyl group of the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2.

[0143] The connection between nucleotides can be:

[0144] a) the phosphate group of the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2 is connected to the hydroxyl group of the overhang structure at the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 1; and / or

[0145] b) the phosphate group of the overhang structure at the 3' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 1 is connected to the hydroxyl group of the overhang structure at the 5' end of the RNA segment of the RNA / DNA hybrid double-stranded fragment 2.

[0146] The DNA in the RNA / DNA hybrid double-stranded fragments can or can not be linked to each other, and in some cases, the DNA can be modified to prevent the linkage of the DNA to each other to form long chain DNA.

[0147] The ligase polypeptide can be a ligase, such as a DNA ligase or an RNA ligase, or a functional fragment of any DNA ligase or RNA ligase, which can be any fragment of the ligase that can perform the ligation function.

[0148] The ligase polypeptide can be ATP-dependent and / or NAD + dependent.

[0149] The ligase polypeptide can be from a eukaryote, a prokaryote, and / or a bacteriophage. The ligase polypeptide can be from a eukaryote, the ligase polypeptide can be from a prokaryote, such as E. coli, and the ligase polypeptide can be from a bacteriophage.

[0150] The ligase polypeptide can include, but is not limited to, one or more of the following groups: T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase, eukaryotic DNA ligase I, eukaryotic DNA ligase III, eukaryotic DNA ligase IV, E. coli DNA ligase, Hi-T4 TM DNA ligase, Salt-T4 DNA ligase, 9°N TM DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase, ligase, thermostable 5' App DNA / RNA Ligase, RtcB ligase, and PBCV DNA ligase. The ligase polypeptide can also include an isozyme of any ligase in the above groups and / or a fragment of any ligase that can perform the ligation function.

[0151] reaction conditions

[0152] The method of making the target single-stranded RNA described herein includes forming a plurality of RNA / DNA hybrid double-stranded fragments and ligating the plurality of RNA / DNA hybrid double-stranded fragments to form a RNA / DNA hybrid double-stranded.

[0153] The temperature for forming the plurality of RNA / DNA hybrid double-stranded fragments can range from about 4°C to about 37°C.

[0154] The temperature range for the formation of the plurality of RNA / DNA hybrid double stranded segments is about 4°C to about 37°C, about 5°C to about 37°C, about 6°C to about 37°C, about 7°C to about 37°C, about 8°C to about 37°C, about 9°C to about 37°C, about 10°C to about 37°C, about 11°C to about 37°C, about 12°C to about 37°C, about 13°C to about 37°C, about 14°C to about 37°C, about 15°C to about 37°C, about 16°C to about 37°C, about 17°C to about 37°C, about 18°C to about 37°C, about 19°C to about 37°C, about 20°C to about 37°C, about 21°C to about 37°C, about 22°C to about 37°C, about 23°C to about 37°C, about 24°C to about 37°C, about 25°C to about 37°C, about 26°C to about 37°C, about 27°C to about 37°C, about 28°C to about 37°C, about 29°C to about 37°C, about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0155] The temperature range for the formation of the plurality of RNA / DNA hybrid double stranded segments is about 4°C to about 10°C, about 4°C to about 20°C, about 4°C to about 30°C, about 4°C to about 37°C, about 10°C to about 20°C, about 10°C to about 30°C, about 10°C to about 37°C, about 20°C to about 30°C, about 20°C to about 37°C, or about 30°C to about 37°C.

[0156] The temperature range for the formation of the plurality of RNA / DNA hybrid double stranded segments is about 4°C to about 5°C, about 4°C to about 6°C, about 4°C to about 7°C, about 4°C to about 8°C, about 4°C to about 9°C, or about 4°C to about 10°C. The temperature range for the formation of the plurality of RNA / DNA hybrid double stranded segments is about 10°C to about 37°C. The temperature range for the formation of the plurality of RNA / DNA hybrid double stranded segments is about 11°C to about 37°C, about 12°C to about 37°C, about 13°C to about 37°C, about 14°C to about 37°C, about 15°C to about 37°C, about 16°C to about 37°C, about 17°C to about 37°C, about 18°C to about 37°C, about 19°C to about 37°C, about 20°C to about 37°C, about 21°C to about 37°C, about 22°C to about 37°C, about 23°C to about 37°C, about 24°C to about 37°C, about 25°C to about 37°C, about 26°C to about 37°C, about 27°C to about 37°C, about 28°C to about 37°C, about 29°C to about 37°C, about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0157] The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 10°C to about 20°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 10°C to about 11°C, about 10°C to about 12°C, about 10°C to about 13°C, about 10°C to about 14°C, about 10°C to about 15°C, about 10°C to about 16°C, about 10°C to about 17°C, about 10°C to about 18°C, or about 10°C to about 19°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is 20°C to about 37°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 20°C to about 37°C, about 21°C to about 37°C, about 22°C to about 37°C, about 23°C to about 37°C, about 24°C to about 37°C, about 25°C to about 37°C, about 26°C to about 37°C, about 27°C to about 37°C, about 28°C to about 37°C, about 29°C to about 37°C, about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0158] The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is 20°C to about 30°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 20°C to about 21°C, about 20°C to about 22°C, about 20°C to about 23°C, about 20°C to about 24°C, about 20°C to about 25°C, about 20°C to about 26°C, about 20°C to about 27°C, about 20°C to about 28°C, or about 20°C to about 29°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 30°C to about 37°C, the temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0159] The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 30°C to about 37°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0160] The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 30°C to about 37°C. The temperature range for forming the plurality of RNA / DNA hybrid double stranded segments is about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0161] The temperature range at which the plurality of RNA / DNA hybrid double stranded segments are connected to form RNA / DNA hybrid double strands is about 4°C to about 37°C, about 5°C to about 37°C, about 6°C to about 37°C, about 7°C to about 37°C, about 8°C to about 37°C, about 9°C to about 37°C, about 10°C to about 37°C, about 11°C to about 37°C, about 12°C to about 37°C, about 13°C to about 37°C, about 14°C to about 37°C, about 15°C to about 37°C, about 16°C to about 37°C, about 17°C to about 37°C, about 18°C to about 37°C, about 19°C to about 37°C, about 20°C to about 37°C, about 21°C to about 37°C, about 22°C to about 37°C, about 23°C to about 37°C, about 24°C to about 37°C, about 25°C to about 37°C, about 26°C to about 37°C, about 27°C to about 37°C, about 28°C to about 37°C, about 29°C to about 37°C, about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0162] The temperature range at which the plurality of RNA / DNA hybrid double stranded segments are connected to form RNA / DNA hybrid double strands is about 4°C to about 10°C, about 4°C to about 20°C, about 4°C to about 30°C, about 4°C to about 37°C, about 10°C to about 20°C, about 10°C to about 30°C, about 10°C to about 37°C, about 20°C to about 30°C, about 20°C to about 37°C, or about 30°C to about 37°C.

[0163] The temperature range at which the plurality of RNA / DNA hybrid double stranded segments are connected to form RNA / DNA hybrid double strands is about 4°C to about 5°C, about 4°C to about 6°C, about 4°C to about 7°C, about 4°C to about 8°C, about 4°C to about 9°C, or about 4°C to about 10°C. The temperature range at which the plurality of RNA / DNA hybrid double stranded segments are connected to form RNA / DNA hybrid double strands is about 10°C to about 37°C. The temperature range at which the plurality of RNA / DNA hybrid double stranded segments are connected to form RNA / DNA hybrid double strands is about 11°C to about 37°C, about 12°C to about 37°C, about 13°C to about 37°C, about 14°C to about 37°C, about 15°C to about 37°C, about 16°C to about 37°C, about 17°C to about 37°C, about 18°C to about 37°C, about 19°C to about 37°C, about 20°C to about 37°C, about 21°C to about 37°C, about 22°C to about 37°C, about 23°C to about 37°C, about 24°C to about 37°C, about 25°C to about 37°C, about 26°C to about 37°C, about 27°C to about 37°C, about 28°C to about 37°C, about 29°C to about 37°C, about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0164] The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 10°C to about 20°C. The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 10°C to about 11°C, about 10°C to about 12°C, about 10°C to about 13°C, about 10°C to about 14°C, about 10°C to about 15°C, about 10°C to about 16°C, about 10°C to about 17°C, about 10°C to about 18°C, or about 10°C to about 19°C. The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is 20°C to about 37°C. The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 20°C to about 37°C, about 21°C to about 37°C, about 22°C to about 37°C, about 23°C to about 37°C, about 24°C to about 37°C, about 25°C to about 37°C, about 26°C to about 37°C, about 27°C to about 37°C, about 28°C to about 37°C, about 29°C to about 37°C, about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0165] The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is 20°C to about 30°C. The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 20°C to about 21°C, about 20°C to about 22°C, about 20°C to about 23°C, about 20°C to about 24°C, about 20°C to about 25°C, about 20°C to about 26°C, about 20°C to about 27°C, about 20°C to about 28°C, or about 20°C to about 29°C. The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 30°C to about 37°C, the temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0166] The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 30°C to about 37°C. The temperature range for said ligating said plurality of RNA / DNA hybrid double stranded segments to form RNA / DNA hybrid double strands is about 30°C to about 37°C, about 31°C to about 37°C, about 32°C to about 37°C, about 33°C to about 37°C, about 34°C to about 37°C, about 35°C to about 37°C, or about 36°C to about 37°C.

[0167] purification method

[0168] The method for preparing the target single-stranded RNA described in the present application can further comprise a purification method to obtain the target single-stranded RNA.

[0169] The forming of the RNA / DNA hybrid duplex or the forming of the RNA / DNA hybrid duplex fragment can further comprise: purifying by reverse-phase HPLC or ion HPLC to obtain the successfully ligated RNA / DNA hybrid duplex.

[0170] The reverse-phase HPLC purification can comprise that the polarity of the stationary phase of the reverse-phase HPLC purification is less than the polarity of the mobile phase.

[0171] The stationary phase of the reverse-phase HPLC purification can be C8 or C18.

[0172] The reverse-phase HPLC purification method can refer to an HPLC purification method for separating oligonucleotide chains (with and without modification groups) of different lengths according to the hydrophobicity of the separated substances. Reverse-phase HPLC is a very effective purification method for purifying target sequences with hydrophobic modification groups and non-target sequences without hydrophobic modification groups. The reverse-phase buffer used in reverse-phase HPLC purification according to the hydrophobicity of the separated substances can use components with different degrees of hydrophobicity.

[0173] The mobile phase can be a combination of reverse-phase buffer A and reverse-phase buffer B, wherein the reverse-phase buffer A can be a solution of triethylamine carbonate (TEAB), triethylamine (TEA), or triethylamine acetate (TEAA).

[0174] The concentration range of the triethylamine carbonate (TEAB), triethylamine (TEA), or triethylamine acetate (TEAA) can be 50-150 mM.

[0175] The reverse-phase buffer B can be an acetonitrile solution of buffer A.

[0176] The flow rate of the mobile phase can be 0.8-1.2 ml / min.

[0177] The purification method, wherein the volume ratio of the reverse-phase buffer B in the mobile phase at 43 minutes before gradient elution can be 10%-40%.

[0178] The ion HPLC purification can comprise that the stationary phase of the ion HPLC purification can be an ion exchange medium.

[0179] The ion HPLC purified stationary phase can be an anion exchange medium. In some embodiments, the ion HPLC purified stationary phase is a weak anion exchange medium (Weak Anion Exchange), preferably weak anion exchange medium DEAE. In some embodiments, the ion HPLC purified stationary phase is a strong anion exchange medium (Strong Anion Exchange), preferably strong anion exchange medium Q.

[0180] The ion HPLC purified stationary phase can be a weak anion exchange medium DEAE.

[0181] The ion HPLC purified stationary phase can be a strong anion exchange medium Q.

[0182] The ion HPLC purification can separate target molecules and non-target molecules by gradient elution according to the amount of negative charge carried by the target molecules. The ion buffer used in the ion HPLC purification can use buffer components with different charges according to the amount of negative charge carried by the target molecules.

[0183] The mobile phase can be a combination of ion buffer A and ion buffer B, wherein the ion buffer A can include urea and phosphate buffer, or urea and TRIS buffer.

[0184] The ion buffer A can include urea and phosphate buffer.

[0185] The ion buffer A can include urea and TRIS buffer.

[0186] The concentration of the urea can be 0-8M.

[0187] The concentration of the phosphate buffer and TRIS buffer can be 50-150mM.

[0188] The ion buffer B can include urea, phosphate buffer or TRIS, guanidine hydrochloride or NaCl solution.

[0189] In some embodiments, the ion buffer B can include urea, phosphate buffer and guanidine hydrochloride solution.

[0190] In some embodiments, the ion buffer B can include urea, phosphate buffer and NaCl solution.

[0191] In some embodiments, the ion buffer B can include urea, TRIS and guanidine hydrochloride solution.

[0192] In some embodiments, the ion buffer B can include urea, TRIS and NaCl solution.

[0193] The flow rate of the mobile phase can be 0.8-1.2 ml / min.

[0194] In some embodiments, the volume ratio of reverse phase buffer B in the mobile phase can be 10%-80% at 63 minutes before gradient elution.

[0195] In the purification method, the purification temperature can be 25-60°C.

[0196] In the purification method, the purification temperature can be 25-40°C, 26-40°C, 27-40°C, 28-40°C, 29-40°C, 30-40°C, 31-40°C, 32-40°C, 33-40°C, 34-40°C, 35-40°C, 36-40°C, 37-40°C, 38-40°C, or 39-40°C.

[0197] In another aspect, the application provides the target single-stranded RNA obtained by the above method.

[0198] In another aspect, the application provides the use of the above method for obtaining the target single-stranded RNA.

[0199] Without being limited by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the application, and are not intended to limit the scope of the application.

[0200] Examples

[0201] Example 1: Efficiency exploration of traditional ligase synthesis method and zigzag connection method

[0202] Using 100 nt of RNA, the traditional ligase synthesis method and the zigzag connection method were compared. The sequence was divided into oligonucleotide chain 1 and oligonucleotide chain 2. According to the traditional ligase synthesis method, the DNA sequence paired with it was designed as oligonucleotide chain 3.

[0203] According to the zigzag connection, the DNA sequence paired with it was designed as oligonucleotide chain 4 and oligonucleotide chain 5, respectively. The sequence is shown in Table 1. The 5' end of oligonucleotide chain 2 was modified with a phosphate group. The 3' and 5' ends of oligonucleotide chain 4 and oligonucleotide chain 5 were modified with an amino group.

[0204] Table 1: Oligonucleotide sequence information

[0205] The specific experimental steps are as follows:

[0206] The traditional ligase synthesis method connection system is as follows:

[0207] Table 2: Traditional ligase synthesis method connection system

[0208] The zigzag connection system is as follows:

[0209] Table 3 zigzag connection system

[0210] After the mixed solution is prepared according to the above system, it is placed in a constant temperature metal bath, and incubated at 25°C for 1 hour. After the reaction is completed, 1 / 10 volume of DNase I and 10x buffer are added, and incubated at 37°C for 15 minutes. After termination, 2x RNA loading buffer is used to terminate the reaction, and denatured at 60°C for 5 minutes. Then Urea-PAGE gel electrophoresis is used for analysis.

[0211] The Urea-PAGE detection result is shown in FIG. 4. Since the secondary structure of this sequence is very strong, the efficiency of the traditional connection method is very low, and the zigzag connection method can effectively connect. Image J software is used for gray scale analysis of the image, and the connection efficiency is calculated as Production gray value / (oligonucleotide chain gray value+Production gray value). The connection efficiency of the zigzag connection method is about 75%, while the efficiency of the traditional connection method is only about 10%. In comparison, the efficiency of the zigzag connection method is significantly higher than that of the traditional connection method.

[0212] Example 2 Influence of DNA modification on zigzag connection method

[0213] In order to compare the influence of DNA modification on the zigzag connection method, the present application uses 50nt RNA to split the sequence into oligonucleotide chain 6 and oligonucleotide chain 7, and designs the DNA sequence paired with them according to the zigzag connection method. The 3' and 5' ends of oligonucleotide chain 8 and oligonucleotide chain 9 are both modified with amino groups, and the 3' and 5' ends of oligonucleotide chain 10 and oligonucleotide chain 11 are both modified with hydroxyl groups.

[0214] Table 4 Oligonucleotide sequence information

[0215] The zigzag connection system is as follows:

[0216] Table 5 zigzag connection system

[0217] The mixed solution prepared according to the above system was placed in a constant temperature metal bath, and incubated at 25°C for 1 hour. After the reaction was completed, 1 / 10 volume of DNase I and 10x buffer were added, and incubated at 37°C for 15 minutes. After termination, 2x RNA loading buffer was used to terminate the reaction, and denatured at 60°C for 5 minutes. Then Urea-PAGE gel electrophoresis was used for analysis.

[0218] The Urea-PAGE detection results are shown in Figure 5. Because the secondary structure of this sequence is strong, the efficiency of traditional ligation is very low, while zigzag ligation can effectively connect. Image J software was used to analyze the gray scale of the image, and the ligation efficiency was calculated as Production gray value / (oligonucleotide chain gray value+Production gray value). The experimental results show that there is no difference between diamino blocked DNA and dihydroxy blocked DNA for zigzag ligation.

[0219] Example 3 Effect of DNA pairing sequences of different lengths on zigzag ligation

[0220] On the basis of the above experiment, DNA pairing sequences of different lengths were designed to test the effect of different length interfaces on the ligation efficiency of zigzag ligation. The sequence information is shown in the following table.

[0221] Table 6 Oligonucleotide sequence information

[0222] Table 7 zigzag ligation system

[0223] The mixed solution prepared according to the above system was placed in a constant temperature metal bath, and incubated at 25°C for 1 hour. After the reaction was completed, 1 / 10 volume of DNase I and 10x buffer were added, and incubated at 37°C for 15 minutes. After termination, 2x RNA loading buffer was used to terminate the reaction, and denatured at 60°C for 5 minutes. Then Urea-PAGE gel electrophoresis was used for analysis.

[0224] The Urea-PAGE detection results are shown in Figure 6. Image J software was used to analyze the gray scale of the image, and the ligation efficiency was calculated as Production gray value / (oligonucleotide chain gray value+Production gray value). The experimental results prove that DNA with different interface lengths of 6-16 nt has no obvious effect on the ligation efficiency of zigzag ligation, and the ligation efficiency is about 75%.

[0225] Example 4 Effect of different reaction temperatures on zigzag ligation

[0226] On the basis of the above experiments, the 10nt interface was selected to test the effect of different reaction temperatures on the connection efficiency of the zigzag connection method. The system was prepared as shown in Table 3.

[0227] After the mixed solution was prepared according to the above system, it was placed in a constant temperature metal bath, and different temperatures 4°C, 10°C, 16°C, 25°C, 37°C were selected for incubation for 1 hour. After the reaction was completed, 1 / 10 volume of DNase I and 10x buffer were added, and incubated at 37°C for 15 minutes. After termination of the reaction with 2x RNA loading buffer, denaturation was performed at 60°C for 5 minutes. Then Urea-PAGE gel electrophoresis was used for analysis.

[0228] The Urea-PAGE detection results are shown in Figure 7. Image J software was used to analyze the gray scale of the image, and the connection efficiency was calculated as Production gray value / (oligonucleotide chain gray value+Production gray value). The experimental results showed that different reaction temperatures from 4-37°C had no obvious effect on the connection efficiency, and the connection efficiency was about 75%.

[0229] Example 5 Effect of different T4 RNA ligases on zigzag connection method

[0230] On the basis of the above experiments, the 10nt interface was selected to test the effect of different reaction temperatures on the connection efficiency of the zigzag connection method. The system was prepared as shown in Table 3.

[0231] Table 8 zigzag connection system

[0232] After the mixed solution was prepared according to the above system, it was placed in a constant temperature metal bath, and different temperatures 4°C, 10°C, 16°C, 25°C, 37°C were selected for incubation for 1 hour. After the reaction was completed, 1 / 10 volume of DNase I and 10x buffer were added, and incubated at 37°C for 15 minutes. After termination of the reaction with 2x RNA loading buffer, denaturation was performed at 60°C for 5 minutes. Then Urea-PAGE gel electrophoresis was used for analysis.

[0233] The Urea-PAGE detection results are shown in Figure 7. Image J software was used to analyze the gray scale of the image, and the connection efficiency was calculated as Production gray value / (oligonucleotide chain gray value+Production gray value). The experimental results proved that after increasing the concentration, there was no difference between the two in the connection efficiency between 0.5μM-4μM, therefore 1μM was selected as the enzyme concentration for zigzag connection.

[0234] Example 6 Specificity of zigzag connection method

[0235] In order to test the specificity of zigzag connection mode, the effect of direct connection of 70 nt RNA oligonucleotide chains with phosphate and hydroxyl modified oligonucleotide chains and double hydroxyl and double phosphate modified oligonucleotide chain connection on zigzag connection was tested by splitting the sequence into oligonucleotide chain 12 and oligonucleotide chain 13 and oligonucleotide chain 14 and oligonucleotide chain 15. Among them, oligonucleotide chain 12 and oligonucleotide chain 13 are both 5' phosphate modified, while oligonucleotide chain 14 is 5' and 3' phosphate modified, and oligonucleotide chain 15 is double hydroxyl modified. According to the zigzag connection design, the DNA sequence paired with it is oligonucleotide chain 16 and oligonucleotide chain 17, respectively, and the sequence is shown in Table 6, and -NH2 modification is added to the 3' end and 5' end of oligonucleotide chain 16 and oligonucleotide chain 17.

[0236] Table 9 Oligonucleotide sequence information

[0237] Wherein m represents that the next nucleotide is methylated, and * represents that the two nucleotides are connected by a phosphorothioate bond.

[0238] The specific experimental steps are as follows:

[0239] The zigzag connection system is as follows:

[0240] Table 10 zigzag connection system

[0241] Table 11 zigzag connection system

[0242] After the mixed solution is prepared according to the above system, it is placed in a constant temperature metal bath, and incubated at 25°C for 1 hour. After the reaction is completed, 1 / 10 volume of DNase I and 10x buffer solution are added, and incubated at 37°C for 15 minutes. After the end, the reaction is terminated with 2x RNA loading buffer, and denatured at 60°C for 5 minutes. Then Urea-PAGE gel electrophoresis is used for analysis.

[0243] The Urea-PAGE detection result is shown in Figure 6. Image J software is used to analyze the gray scale of the image, and the connection efficiency is calculated as Production gray value / (oligonucleotide chain gray value+Production gray value). Different modified oligonucleotide chains have no by-products through zigzag connection.

[0244] Example 7 Optimization of zigzag purification method

[0245] In view of the particularity of zigzag connection mode, the present application designs two strategies of reverse phase HPLC purification and ion HPLC purification respectively, and the purification core is to open the hydrogen bond between 6-16 bases on the sticky end between the two fragments that have not been connected by weak denaturation force, while the hydrogen bond on the whole chain after connection does not break, so that by separating double-stranded of different lengths, the connection product and the two fragments that have not been completely reacted can be effectively separated, and the performance on Urea-PAGE is that the connection product band has two Adaptor bands below.

[0246] The experimental steps of the reverse phase purification method development are as follows:

[0247] Table 12 Reverse phase purification basic information

[0248] Preparation of mobile phase for reverse phase purification:

[0249] Buffer A (reverse phase): 100 mM TEAB in H2O (pH 7.0)

[0250] Prepare 2M TEAB stock solution: for example, accurately measure 139.19 mL of triethylamine in a 500 mL system of 2M TEAB, add ultrapure water to 500 mL, shake well, and then charge CO2 to the solution to pH 7.0 (Table 13).

[0251] Table 13 2M TEAB (pH 7.0) preparation system

[0252] For example, take 1L of buffer A (reverse phase) as an example, accurately measure 50 mL of 2M TEAB stock solution with a 1L measuring cylinder, add ultrapure water to 1L, and store at room temperature. The appropriate preparation volume can be selected according to the required amount (Table 14).

[0253] Table 14 Buffer A (reverse phase) preparation system

[0254] Buffer B (reverse phase): 50% buffer A in ACN (chromatographic acetonitrile) (pH 7.0)

[0255] For example, take 1L of buffer B (reverse phase) as an example, accurately measure 500 mL of buffer A (reverse phase) with a 1L measuring cylinder, add chromatographic acetonitrile to 1L, and store at room temperature. The appropriate preparation volume can be selected according to the required amount (Table 15).

[0256] Table 15 Buffer B (reverse phase) preparation system

[0257] After the above experimental conditions are prepared, the following purification conditions are selected to carry out reverse phase purification of zigzag connection crude product (Table 16).

[0258] Table 16 Reverse phase purification gradient

[0259] After HPLC purification, according to the different peaks presented on the chromatogram, the corresponding fractions were recovered and analyzed by Urea-PAGE, as shown in Figures 10 and 11.

[0260] As shown in Figures 10 and 11, the zigzag connection crude product was first completely separated from the two incomplete reaction fragments, referred to as "P1 and P2", under the reverse phase purification conditions, i.e. the positions corresponding to D3 on the HPLC and PAGE charts. In addition, the subsequent separation was also for the separation of the two adaptors. The final F5 fraction was the connection product with two adaptors, which fully met the expected separation target. In this way, the effective separation of the two double-stranded incomplete connection and the target double-stranded successful connection in the enzyme connection system was achieved.

[0261] The ion purification method development experiment steps are as follows:

[0262] Table 17 Ion purification basic information

[0263] Ion purification mobile phase preparation:

[0264] Buffer A (ion): 75mM PBS + 2M urea + 10% ACN

[0265] For example, take 1L of buffer A, accurately weigh 120g of urea, 4.56g of sodium dihydrogen phosphate dihydrate and 16.4g of disodium hydrogen phosphate dodecahydrate, add 850mL of ultrapure water and completely dissolve under ultrasonic action, measure and dissolve to 900mL, filter the dissolved mobile phase with a 0.45μm filter membrane, after filtration, add 100mL of chromatographic acetonitrile to make up to 1L, after preparation, ultrasonic for 10 minutes at room temperature, then get buffer A, put it in a blue cap bottle and label it for room temperature storage (Table 18).

[0266] Table 18 Buffer A (ion) preparation system

[0267] Buffer B (ion): 75mM PBS + 1M guanidine hydrochloride + 2M urea + 10% ACN

[0268] Take 1 L beaker as an example, accurately weigh 120 g urea, 95.5 g guanidine hydrochloride, 4.56 g sodium dihydrogen phosphate dihydrate and 16.4 g disodium hydrogen phosphate dodecahydrate, add 850 mL ultrapure water to completely dissolve under ultrasonic action, measure to 900 mL, filter the dissolved mobile phase with a 0.45 μm filter membrane, after filtration, add 100 mL chromatographic acetonitrile to 1 L, after preparation, ultrasonic for 10 minutes at room temperature, and then buffer B is obtained, which is placed in a blue cap bottle and labeled for room temperature storage (Table 19).

[0269] Table 19 Buffer B (ion) preparation system

[0270] After the above experimental conditions are prepared, the following purification conditions are selected to carry out zigzag connection crude ion purification (Table 20).

[0271] Table 20 Ion purification gradient

[0272] After HPLC purification, according to the different peaks presented on the chromatogram, the corresponding components are recovered and subjected to Urea-PAGE detection analysis, as shown in Figures 12 and 13.

[0273] As shown in Figures 12 and 13, the zigzag connection crude under the ion purification conditions shows a significant difference in HPLC chromatogram from the reverse phase purification due to the different separation modes. From the PAGE diagram, it can be seen that when ion purification separation is carried out, two Adaptors are preferentially separated first, as shown in positions B2 and B9, and the key separation target “P1 and P2” is completely separated with part of the two Adaptors and located near the main peak, as shown in C7. However, the finally recovered C12 and D1 components are the connection products with two Adaptors, which also meet the expected separation target. Therefore, under the ion purification conditions, the two double-stranded incomplete connection and the target double-stranded successful connection in the enzyme connection system can still be effectively separated.

[0274] In summary, the reverse phase and ion purification strategies developed in the present application can both achieve effective separation of the target product of the zigzag connection system.

[0275] Example 8 Influence of different RNA ligases on zigzag connection mode

[0276] We compared the ligation efficiency of T4 RNA ligase2, Anc-T4-2 and RTCB. We split the 94nt RNA sequence into Oligo 18 and Oligo 19, where the 5’ and 3’ end groups of oligo 18 are both hydroxyl, and the 5’ and 3’ end groups of Oligo 19 are both phosphate modified. The DNA sequence Oligo 20 and Oligo 21 are designed to pair with them according to zigzag ligation. This sequence is suitable for T4 RNA ligase2, Anc-T4-2. Since RTCB ligates the 5’ hydroxyl and 3’ phosphate of RNA, we split the 94nt RNA sequence into Oligo 22 and Oligo 23 in the same way, where the 5’ and 3’ end groups of oligo 22 are both phosphate modified, and the 5’ and 3’ end groups of Oligo 23 are both hydroxyl. The above ligation sequences with modified groups were subjected to two-step enzymatic ligation, and the ligation method is referred to Example 5. The Page detection results show that the ligation efficiency of T4 RNA ligase2 is not much different from that of RTCB and Anc-T4-2 (Figure 14).

[0277] Finally we compared the ligation efficiency of T4 RNA ligase2 and R2D. We split the 100nt RNA sequence into Oligo 26 and Oligo 27, where the 5’ and 3’ end groups of oligo 26 are both hydroxyl, and the 5’ and 3’ end groups of Oligo 27 are both phosphate modified. The DNA sequence Oligo 28 and Oligo 29 are designed to pair with them according to zigzag ligation. The above ligation sequences with modified groups were subjected to two-step enzymatic ligation, and the ligation method is referred to Example 5. The Page detection results show that the ligation efficiency of T4 RNA ligase2 is not much different from that of R2D (Figure 15).

[0278] Table 21

Claims

1. A method for preparing target single-stranded RNA, comprising: (1) Provide multiple RNA fragments based on the target single-stranded RNA; (2) Based on the plurality of RNA fragments, a plurality of DNA fragments are provided to form a plurality of RNA / DNA hybrid double-stranded fragments, wherein the DNA fragments and the corresponding RNA fragments form RNA / DNA hybrid double-stranded fragments, wherein the RNA / DNA hybrid double-stranded fragments have complementary base regions, and one or both ends of the RNA / DNA hybrid double-stranded fragments have protruding structures; (3) In the presence of a ligase polypeptide, the plurality of RNA / DNA hybrid double-stranded fragments are ligated to form an RNA / DNA hybrid double strand, wherein the RNA / DNA hybrid double strand includes the target single-stranded RNA. (4) Remove the DNA strand or DNA fragment from the RNA / DNA hybrid double strand to obtain the target single-stranded RNA.

2. The method according to claim 1, wherein the length of the RNA fragment is 20-10000 nt.

3. The method according to any one of claims 1-2, wherein the length of the protruding structure is 4-20 nt.

4. The method according to any one of claims 1-3, wherein the plurality of RNA / DNA hybrid double-stranded fragments comprises RNA / DNA hybrid double-stranded fragment 1 and an adjacent RNA / DNA hybrid double-stranded fragment 2, wherein the 3' end of the RNA fragment of RNA / DNA hybrid double-stranded fragment 1 and the 5' end of the RNA fragment of RNA / DNA hybrid double-stranded fragment 2 are joined to form a continuous portion of the target single-stranded RNA.

5. The method according to claim 4, wherein the prominent structure of the RNA / DNA hybrid double-stranded fragment 1 is base-complementary to the prominent structure of the RNA / DNA hybrid double-stranded fragment 2.

6. The method according to claims 4-5, wherein a) The 5' protrusion of the DNA fragment in RNA / DNA hybrid double-stranded segment 1 is complementary to the bases of the 5' protrusion of the RNA fragment in RNA / DNA hybrid double-stranded segment 2; or b) The protruding structure at the 3' end of the RNA fragment in RNA / DNA hybrid double-stranded segment 1 is complementary to the protruding structure at the 3' end of the DNA fragment in RNA / DNA hybrid double-stranded segment 2.

7. The method according to any one of claims 4-6, wherein the 3' end of the RNA fragment of the RNA / DNA hybrid double-stranded fragment 1 is modified with a hydroxyl group or a phosphate group.

8. The method according to any one of claims 4-7, wherein the 5' end of the RNA fragment of the RNA / DNA hybrid double-stranded fragment 2 is modified with phosphate or hydroxyl groups.

9. The method according to any one of claims 1-8, wherein the DNA fragment has no modification or has hydroxyl modification at both ends.

10. The method according to any one of claims 1-8, wherein the 5' end and / or 3' end of the DNA comprises amino modification.

11. The method according to any one of claims 1-10, wherein the plurality of RNA / DNA hybrid double-stranded fragments comprises an RNA / DNA hybrid double-stranded 5' end fragment and / or an RNA / DNA hybrid double-stranded 3' end fragment, wherein the RNA fragment of the RNA / DNA hybrid double-stranded 5' end fragment has a blunt end at 5', and the RNA fragment of the RNA / DNA hybrid double-stranded 3' end fragment has a blunt end at 3'.

12. The method according to any one of claims 1-11, wherein the ligase polypeptide comprises an RNA ligase polypeptide.

13. The method according to claim 12, wherein the RNA ligase polypeptide comprises T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2, T3 DNA ligase, T7 DNA ligase, eukaryotic DNA ligase I, eukaryotic DNA ligase III, eukaryotic DNA ligase IV, E. coli DNA ligase, or Hi-T4. TM DNA ligase, Salt-T4 DNA ligase, 9°NTM DNA ligase, Taq DNA ligase, HiFi Taq DNA ligase One or more of the following: ligase, thermostable 5' App DNA / RNA ligase, RtcB ligase, and PBCV DNA ligase.

14. The method according to any one of claims 1-13, wherein step (4) further comprises: In the presence of DNase, the DNA strand or DNA fragment in the RNA / DNA hybrid double strand is removed to obtain the target single-stranded RNA.

15. The method according to any one of claims 1-14, further comprising: Based on the target single-stranded RNA, N RNA fragments are provided, wherein the N RNA fragments are linked together to form the complete target single-stranded RNA.

16. The method of claim 15, wherein N is an integer greater than or equal to 2.

17. The method according to any one of claims 15-16, further comprising providing N DNA fragments, wherein the N DNA fragments correspond one-to-one with the N RNA fragments to form N RNA / DNA hybrid double-stranded fragments.

18. The method according to any one of claims 1-17, wherein the CG content of the RNA fragment is between 0-100%.

19. The method according to any one of claims 1-18, wherein the protruding structure is a freeline structure with no secondary structures present.

20. The method according to any one of claims 1-19, wherein the temperature range for forming the plurality of RNA / DNA hybrid double-stranded fragments is 4°C-37°C.

21. The method according to any one of claims 1-20, wherein the temperature range for connecting the plurality of RNA / DNA hybrid double-stranded fragments to form an RNA / DNA hybrid double strand is 4°C-37°C.

22. The method according to any one of claims 1-21, wherein forming an RNA / DNA hybrid double strand or forming an RNA / DNA hybrid double strand fragment further comprises: Successfully ligated RNA / DNA hybrid double strands were obtained by purification using reversed-phase HPLC or ion HPLC.

23. The method of claim 22, wherein the reversed-phase HPLC purification comprises that the polarity of the stationary phase in the reversed-phase HPLC purification is less than that of the mobile phase.

24. The method according to claim 23, wherein the stationary phase for reversed-phase HPLC purification is C8 or C18.

25. The method according to any one of claims 23-24, wherein the mobile phase is a combination of reversed-phase buffer A and reversed-phase buffer B, wherein reversed-phase buffer A is a solution of triethylamine carbonate (TEAB), triethylamine (TEA), or triethylamine acetate (TEAA).

26. The method according to claim 25, wherein the concentration of triethylamine carbonate (TEAB), triethylamine (TEA), or triethylamine acetate (TEAA) is in the range of 50-150 mM.

27. The method of claim 26, wherein the reversed-phase buffer B is an acetonitrile solution of buffer A.

28. The method according to any one of claims 23-26, wherein the flow rate of the mobile phase is 0.8-1.2 ml / min.

29. The method according to any one of claims 23-28, wherein the volume ratio of reversed-phase buffer B in the mobile phase is 10%-40% at 43 minutes prior to gradient elution.

30. The method according to claim 22, wherein the ion HPLC purification comprises using an ion exchange medium as the stationary phase for ion HPLC purification.

31. The method according to claim 30, wherein the stationary phase for ion HPLC purification is an anion exchange medium.

32. The method according to claim 31, wherein the stationary phase for ion HPLC purification is a weak anion exchange medium, preferably a weak anion exchange medium DEAE.

33. The method according to claim 31, wherein the stationary phase for ion HPLC purification is a strong anion exchange medium, preferably a strong anion exchange medium Q.

34. The method according to any one of claims 30-33, wherein the mobile phase is a combination of ion buffer A and ion buffer B, wherein ion buffer A comprises urea and phosphate buffer, or urea and TRIS buffer.

35. The method according to claim 34, wherein the concentration of urea is 0-8M.

36. The method according to claim 34, wherein the concentrations of the phosphate buffer and the TRIS buffer are 50-150 mM.

37. The method of claim 34, wherein the ion buffer B comprises urea, phosphate buffer or TRIS, guanidine hydrochloride or NaCl solution.

38. The method according to any one of claims 30-37, wherein the flow rate of the mobile phase is 0.8-1.2 ml / min.

39. The method according to any one of claims 30-38, wherein the volume ratio of reversed-phase buffer B in the mobile phase is 10%-80% at 63 minutes prior to gradient elution.

40. The method according to any one of claims 22-39, wherein the purification temperature is 25°C-60°C.

41. The target single-stranded RNA obtained by the method according to any one of claims 1-39.

42. Use of the method according to any one of claims 1-39 for obtaining the target single-stranded RNA.

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