RNA containing phosphorothioate bond and preparation method therefor

Abstract

Provided are an RNA containing a thioester bond and a preparation method therefor. The method comprises: connecting a 3' hydroxyl group of a substrate with a 5' thiophosphoric acid by using an RNA ligase to form an RNA containing a thioester bond. The 5' thiophosphoric acid comprises a thio-modified 5' phosphoric acid. The thio-modification comprises P-OH of 5' phosphoric acid or the oxygen atom in P=O being replaced by a sulfur atom, correspondingly becoming P-SH or P=S. The RNA ligase comprises any one or more enzymes in RNA ligase family 1, 2 or 3; in particular being one or more of the RNA ligase as shown in SEQ ID NO: 1-22 etc.

Description

RNA containing phosphorothioate bonds and preparation method thereof

[0001] This application is based on the Chinese application with CN application number 202311335112.8 and application date October 16, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of RNA preparation, and in particular to an RNA containing a phosphorothioate bond and a preparation method thereof. Background Art

[0003] RNA drugs are a new class of drugs that have emerged in recent years. They are categorized into three groups based on their mechanism of action: First, small nucleic acid drugs that target nucleic acids and inhibit or activate gene expression, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA), and small activating RNA (saRNA); second, RNA aptamers that target proteins and modulate their activity; and third, messenger RNA (mRNA) that encodes therapeutic proteins or antigens. RNA drugs offer numerous advantages, including strong targeting, simplified design, short development cycles, and a wealth of candidate targets. Since the US FDA approved the first RNA drug, fomivirsen, in 1998 (since withdrawn from the market), dozens of RNA drugs are now commercially available, with many more in development and clinical trials.

[0004] Stability-enhancing chemical modification is a key strategy in the design of nucleic acid drugs. Safety and efficacy are major challenges in the design and development of RNA-targeted drugs and are also the main reasons for early drug development failures. Thio-modification of oligonucleotide phosphodiester bonds—the replacement of the oxygen atom in the P-OH or P=O of the internucleotide phosphodiester bond with a sulfur atom—has been widely used in the development of RNA drugs. Thio-modification of RNA drug phosphodiester bonds has the dual effects of increasing nuclease resistance and promoting binding to plasma proteins, thereby prolonging the drug's elimination half-life, increasing its in vivo circulation time, and improving its pharmacokinetics. Simultaneously, thio-modification introduces chirality, resulting in significantly different pharmacokinetic and pharmacodynamic properties for the two orientations, impacting drug development. Thio-modification can also reduce the affinity of oligonucleotides for their targets. However, prior art syntheses of thio-modified oligonucleotides primarily rely on solid-phase synthesis using chemical reactions using thio reagents. There is no research on the preparation of RNA containing phosphorothioate bonds using biological methods such as enzyme catalysis.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide an RNA containing a phosphorothioate bond and a preparation method thereof, so as to solve the problem in the prior art that it is difficult to prepare RNA containing a phosphorothioate bond using an enzyme catalysis method.

[0007] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for preparing RNA containing a phosphorothioate bond is provided, the method comprising: using RNA ligase to catalyze the connection between the 3′ hydroxyl group of a substrate and a 5′ thiophosphate to form RNA containing a phosphorothioate bond; the 5′ thiophosphate includes a 5′ phosphate containing a thio modification, wherein the thio modification includes replacing the oxygen atom in the P-OH or P═O of the 5′ phosphate with a sulfur atom, correspondingly becoming P-SH or P═S; and any one or more enzymes of RNA ligase families 1, 2, or 3.

[0008] Furthermore, the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; or An enzyme having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or even 99.9% or more identity with any of the RNA ligases shown in NO:22, and a protein having activity of catalyzing the ligation of a 3' hydroxyl group to a 5' thiophosphate.

[0009] Furthermore, the substrate includes single-stranded RNA or double-stranded RNA, and accordingly, the RNA containing phosphorothioate bonds includes single-stranded RNA containing phosphorothioate bonds or double-stranded RNA containing phosphorothioate bonds; the substrate includes 1, 2 or more.

[0010] Furthermore, when there is one substrate and the substrate is single-stranded RNA, the 5′ end of the substrate is a 5′ thiophosphate and the 3′ end of the substrate is a 3′ hydroxyl group. The 5′ end and the 3′ end of the substrate are connected and cyclized under the catalysis of RNA ligase to obtain a cyclic single-stranded RNA containing a thiophosphate bond.

[0011] Furthermore, the splint is used to guide the circularization of the single-stranded RNA, the splint specifically binds to at least three bases from the 3′ end to the 5′ end of the single-stranded RNA, the splint specifically binds to at least three bases from the 5′ end to the 3′ end of the single-stranded RNA, and the base on the splint that specifically binds to the 3′ end of the single-stranded RNA is adjacent to the base that specifically binds to the 5′ end of the single-stranded RNA.

[0012] Furthermore, when there are two substrates and the substrates are single-stranded RNA, the substrates include a first substrate and a second substrate, the 3′ end of the first substrate is a 3′ hydroxyl group, and the 5′ end of the second substrate is a 5′ thiophosphate. The 3′ end of the first substrate and the 5′ end of the second substrate are connected under the catalysis of RNA ligase to obtain a linear single-stranded RNA containing a thiophosphate bond.

[0013] Furthermore, the 3' end of the second substrate is a 3' protecting group, and the 5' end of the first substrate is a 5' protecting group.

[0014] Furthermore, when there are two substrates and the substrate is double-stranded RNA, the substrate includes a sense strand and an antisense strand, and the substrate includes a first substrate and a second substrate; the 3′ end of the sense strand of the first substrate is a 3′ hydroxyl group, and the 5′ end of the antisense strand of the first substrate is a 5′ phosphate group; the 5′ end of the sense strand of the second substrate is a 5′ phosphate group, and the 3′ end of the antisense strand of the second substrate is a 3′ hydroxyl group; at least one of the 5′ phosphate groups of the antisense strand of the first substrate and the sense strand of the second substrate is a 5′ thiophosphate; the 3′ end of the sense strand of the first substrate and the 5′ end of the sense strand of the second substrate are connected under the catalysis of RNA ligase; the 5′ end of the antisense strand of the first substrate and the 3′ end of the antisense strand of the second substrate are connected under the catalysis of RNA ligase; and a linear double-stranded RNA containing a thiophosphate bond is obtained.

[0015] Furthermore, the 3' ends of the antisense strand of the first substrate and the sense strand of the second substrate are both 3' protecting groups; the 5' ends of the sense strand of the first substrate and the 5' ends of the antisense strand of the second substrate are both 5' protecting groups.

[0016] Furthermore, the first substrate and the second substrate are both double-stranded RNAs with different lengths of sense strands and antisense strands, and the 3′ end of the sense strand of the first substrate is complementary paired with the 3′ end of the antisense strand of the second substrate, or the 5′ end of the antisense strand of the first substrate is complementary paired with the 5′ end of the sense strand of the second substrate, so that the 3′ end of the sense strand of the first substrate is adjacent to the 5′ end of the sense strand of the second substrate, and the 5′ end of the antisense strand of the first substrate is adjacent to the 3′ end of the antisense strand of the second substrate.

[0017] Furthermore, the preparation method also includes using a splint to guide the connection between the first substrate and the second substrate; the splint specifically binds to at least 3 bases from the 3' end to the 5' end of the first substrate, and the splint specifically binds to at least 3 bases from the 5' end to the 3' end of the second substrate.

[0018] Furthermore, the base on the splint that specifically binds to the 3' end of the first substrate is adjacent to the base that specifically binds to the 5' end of the second substrate.

[0019] Furthermore, the preparation method is repeatedly used to connect three or more substrates multiple times, with two substrates connected each time, and RNA containing phosphorothioate bonds is obtained by connecting them in sequence.

[0020] Furthermore, the preparation method includes using a splint to guide the connection of three or more substrates; the splint and the substrate both have at least three base-specific bindings.

[0021] Furthermore, the bases of the substrates specifically bound to the splint are adjacent, and the adjacent bases are the 5' end and 3' end of different substrates, respectively. The 5' end is a 5' phosphate, at least one of the 5' phosphates is a 5' thiophosphate; and the 3' end is a hydroxyl group.

[0022] Furthermore, the catalytic system of RNA ligase includes: 1-20000 μM of the substrate, 1-100000 μM ATP, 5-100 mM Tris-HCl, 0.1-20 mM divalent metal ions, 0-10 mM dithiothreitol, and 0.001-10 mg / mL RNA ligase.

[0023] Furthermore, the catalytic temperature of the RNA ligase is 4-50° C., and the catalytic time is 0.1-24 h; and the divalent metal ion is derived from MgCl 2 or MgSO 4 .

[0024] Furthermore, the substrate includes one or more non-natural nucleotides, and the RNA containing phosphorothioate bonds includes non-natural RNA containing phosphorothioate bonds.

[0025] Furthermore, the number of non-natural nucleotides in the substrate is ≥2, and the 5′ end and / or 3′ end of the substrate are non-natural nucleotides.

[0026] Furthermore, the substrate consists of non-natural nucleotides.

[0027] Furthermore, non-natural nucleotides include: ribonucleotides with one or more of the following modifications: ribose 2′ position modification, ribose backbone modification, base modification or phosphate backbone modification; ribose 2′ position modification includes 2′-methoxy modification, 2′-fluorine modification, 2′-hydrogen modification, 2′-methoxyethyl modification, 2′-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; ribose backbone modification includes replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA or dXNA; base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; phosphate backbone modification includes PS modification.

[0028] In order to achieve the above object, according to a second aspect of the present invention, an RNA containing a phosphorothioate bond is provided. The RNA containing a phosphorothioate bond includes the RNA containing a phosphorothioate bond prepared by the above preparation method.

[0029] By applying the technical solution of the present invention and utilizing the above-mentioned preparation method and RNA ligase, the 3′ hydroxyl group of the substrate is connected to the 5′ thiophosphate, and one substrate can be connected into a circular RNA or multiple substrates can be connected into linear RNA, thereby preparing RNA containing thiophosphate bonds that is difficult to obtain in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0031] Figure 1 shows a schematic diagram of a preparation method according to an embodiment of the present invention, wherein Figure 1a shows a schematic diagram of connecting two single-stranded RNAs using the above method; Figure 1b shows a schematic diagram of using a splint to guide the connection of two single-stranded RNA substrates using the above preparation method; Figure 1c shows a schematic diagram of connecting two double-stranded RNAs using the above preparation method; Figure 1d shows a schematic diagram of using the above preparation method to cyclize substrates to form single-stranded circular RNA.

[0032] FIG2 shows a gel electrophoresis diagram according to Example 1 of the present invention.

[0033] FIG3 shows a gel electrophoresis diagram according to Example 2 of the present invention.

[0034] FIG4 shows a UPLC chromatogram according to Example 2 of the present invention.

[0035] FIG5 shows a UPLC chromatogram according to Example 2 of the present invention.

[0036] FIG6 shows a UPLC chromatogram according to Example 3 of the present invention.

[0037] FIG. 7 shows a mass spectrum according to Example 3 of the present invention.

[0038] FIG8 shows a UPLC chromatogram according to Example 4 of the present invention.

[0039] FIG9 shows a mass spectrum according to Example 4 of the present invention.

[0040] FIG10 shows a UPLC chromatogram according to Example 5 of the present invention.

[0041] FIG11 shows a gel electrophoresis diagram according to Example 6 of the present invention. DETAILED DESCRIPTION

[0042] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0043] As mentioned in the background art, there is no prior art research on the use of biological methods such as enzyme catalysis to prepare RNA containing thiophosphate bonds. Different RNA ligases have different requirements for the length, modification, and single and double strands of the ligation substrates. In particular, when catalyzing the ligation of difficult substrates such as those containing thiophosphates or non-natural nucleotides, there are problems such as low catalytic activity and incorrect product ligation. In this application, the inventors attempt to develop a method for preparing RNA containing thiophosphate bonds using RNA ligase, and thus propose a series of protection schemes for this application.

[0044] In a first typical embodiment of the present application, a method for preparing RNA containing a thiophosphate bond is provided, the preparation method comprising: using an RNA ligase to catalyze the connection between the 3' hydroxyl group of a substrate and a 5' thiophosphate to form an RNA containing a thiophosphate bond; the 5' thiophosphate includes a 5' phosphate containing a thio modification, and the thio modification includes that the oxygen atom of P-OH or P=O in the 5' phosphate is replaced by a sulfur atom, correspondingly becoming P-SH or P=S; any one or more enzymes of RNA ligase family 1, 2 or 3; the RNA ligase includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; or an enzyme having 70% or more identity, preferably 75% or more identity, 80% or more identity, 85% or more identity, 90% or more identity, 95% or more identity, 98% or more identity, 99% or more identity, 99.5% or more identity, or 99.9% or more identity to any one of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 22, and having activity of catalyzing the ligation of a 3' hydroxyl group to a 5' thiophosphate.

[0045] The above-mentioned RNA ligase can be used to catalyze the connection between the 3' hydroxyl group and the 5' thiophosphate to form a thiophosphate bond similar to a phosphodiester bond, thereby preparing RNA containing a thiophosphate bond. In this application, 5' (5' end) phosphate / phosphate group / phosphate root refers to the phosphate group on the 5th C atom of the nucleotide at the 5' end of the nucleic acid chain. 5' phosphothioate modification includes 5' thio modification to 5' phosphate P-OH or P=O where the oxygen atom is replaced by a sulfur atom and correspondingly becomes P-SH or P=S, and does not specifically refer to P-SH or specifically P=S. The structure of 5' phosphothioate modification includes One or more of .

[0046] SEQ ID NO: 1 (ligase-1, Escherichia phage T4, RNA ligase 1 (RNA ligase family 1, Rnl1)):

[0047] SEQ ID NO: 2 (ligase-2, Salmonella phage STP4-a, RNA ligase 2 (RNA ligase family 2, Rnl2)):

[0048] SEQ ID NO: 3 (ligase-3, Escherichia phage T4, RNA ligase 2):

[0049] SEQ ID NO: 4 (ligase-4, Vibrio phage KVP40, RNA ligase 2):

[0050] SEQ ID NO: 5 (ligase-5, Yersinia phage fPS-2, RNA ligase 2):

[0051] SEQ ID NO: 6 (ligase-6, Vibrio phage nt-1, RNA ligase 2):

[0052] SEQ ID NO: 7 (ligase-7, Shigella phage Sf22, RNA ligase 1):

[0053] SEQ ID NO:8(ligase-8,Vibrio phage VH7D,RNA ligase 1):

[0054] SEQ ID NO:9(ligase-9,Bacteriophage dhaeg,RNA ligase 2):

[0055] SEQ ID NO:10(ligase-10,Klebsiella phage KP27,RNA ligase 2):

[0056] SEQ ID NO:11(ligase-11,Vibrio phage V05,RNA ligase 1):

[0057] SEQ ID NO:12(ligase-12,Rhodothermuus phage RM378,RNA ligase 1):

[0058] SEQ ID NO:13(ligase-13,Citrobacter phage CkP1,RNA ligase 1):

[0059] SEQ ID NO:14(ligase-14,Escherichia phage JN02,RNA ligase 1):

[0060] SEQ ID NO:15(ligase-15,Klebsiella phage KP179,RNA ligase 1):

[0061] SEQ ID NO:16(ligase-16,Vibrio phage nt-1,RNA ligase 1):

[0062] SEQ ID NO:17(ligase-17,Vibrio phage VH7D,RNA ligase 2):

[0063] SEQ ID NO: 18 (ligase-18, Klebsiella phage KP15, RNA ligase 2):

[0064] SEQ ID NO: 19 (ligase-19, Vibrio phage JN02, RNA ligase 2):

[0065] SEQ ID NO: 20 (ligase-20, Escherichia phage JS98, RNA ligase 2):

[0066] SEQ ID NO: 21 (ligase-21, Escherichia phage JS98, RNA ligase 3 (RNA ligase family 3, Rnl3)):

[0067] SEQ ID NO: 22 (ligase-22, fungus Deinococcus radiodurans, RNA ligase 2):

[0068] Identity as used herein refers to the "identity" between amino acid sequences or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides in an amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0069] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the sequence in a).

[0070] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0071] Substitution and replacement rules generally state that amino acids with similar properties will have similar effects after substitution. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0072] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0073] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0074] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0075] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0076] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0077] In a preferred embodiment, the substrate comprises single-stranded RNA or double-stranded RNA, and accordingly, the RNA containing phosphorothioate bonds comprises single-stranded RNA containing phosphorothioate bonds or double-stranded RNA containing phosphorothioate bonds; the substrate comprises 1, 2 or more.

[0078] The length of the substrate is ≥ 2 nt, including but not limited to 2, 5, 10, 15, 20, 30, 40, 50, 70, 100, 150, 200, 300, 500, 1000, 2000, and 5000 nt. Linking can still be performed with increasing chain length, although the linking efficiency will be reduced to a certain extent.

[0079] The number of substrates used in the above preparation method includes 1 or 2. When the number of substrates is 1, the 3' end of the substrate is connected to a hydroxyl group and the 5' end is connected to a thiophosphate. Using the above preparation method and RNA ligase, the 3' hydroxyl group of the substrate and the 5' thiophosphate can be connected, causing the substrate to cyclize itself to form RNA containing a thiophosphate bond. When the number of substrates is 2, the substrates include a first substrate and a second substrate, the 3' end of the first substrate is connected to a hydroxyl group and the 5' end of the second substrate is connected to a thiophosphate. Using the above preparation method and RNA ligase, the 3' end of the first substrate and the 5' end of the second substrate can be connected via a thiophosphate bond to form RNA containing a thiophosphate bond.

[0080] In a preferred embodiment, when there is one substrate and the substrate is single-stranded RNA, the 5′ end of the substrate is a 5′ phosphorothioate, and the 3′ end of the substrate is a 3′ hydroxyl group. The 5′ end of the substrate and the 3′ end of the substrate are connected and cyclized under the catalysis of RNA ligase to obtain a cyclic single-stranded RNA containing a phosphorothioate bond; preferably, a splint is used to guide the cyclization of the single-stranded RNA, the splint specifically binds to at least 3 bases from the 3′ end to the 5′ end of the single-stranded RNA, the splint specifically binds to at least 3 bases from the 5′ end to the 3′ end of the single-stranded RNA, and the base on the splint that specifically binds to the 3′ end of the single-stranded RNA is adjacent to the base that specifically binds to the 5′ end of the single-stranded RNA.

[0081] The above preparation method can be used to connect the 5′ and 3′ ends of the substrate end-to-end to form a single-stranded circular RNA. The preparation of the single-stranded circular RNA also includes the use of a splint to guide the circularization of the single-stranded RNA. The splint can specifically bind to the bases at the 5′ and 3′ ends of the substrate single-stranded RNA, thereby reducing the spatial distance between the 5′ and 3′ ends and facilitating ligation by RNA ligase.

[0082] The above-mentioned RNA ligase includes, but is not limited to, any one or more of ligase-1 (SEQ ID NO: 1), ligase-4 (SEQ ID NO: 4), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-12 (SEQ ID NO: 12), ligase-13 (SEQ ID NO: 13), ligase-14 (SEQ ID NO: 14), ligase-15 (SEQ ID NO: 15), ligase-16 (SEQ ID NO: 16), ligase-18 (SEQ ID NO: 18), and ligase-19 (SEQ ID NO: 19).

[0083] In a preferred embodiment, when there are two substrates and the substrates are single-stranded RNA, the substrates include a first substrate and a second substrate, the 3′ end of the first substrate is a 3′ hydroxyl group, and the 5′ end of the second substrate is a 5′ thiophosphate. The 3′ end of the first substrate and the 5′ end of the second substrate are connected under the catalysis of RNA ligase to obtain a linear single-stranded RNA containing a thiophosphate bond; preferably, the 3′ end of the second substrate is a 3′ protecting group, and the 5′ end of the first substrate is a 5′ protecting group.

[0084] Using the above preparation method, the first substrate and the second substrate can be connected to form a single-stranded linear RNA. The above 5' protecting group includes but is not limited to a hydroxyl group, an N-acetylgalactosamine group, -O-NH2, an azido group, a cyanoethyl group, an allyl group, or a 2-nitrobenzyl group. The above 3' protecting group includes but is not limited to a phosphate group, an N-acetylgalactosamine group, -O-NH2, an azido group, a cyanoethyl group, an allyl group, or a 2-nitrobenzyl group. In order to prevent the substrate from cyclizing itself, a single substrate cannot have both a 5'thiophosphate and a 3' terminal hydroxyl group, that is, the above 3' protecting group does not need to be a hydroxyl group, and the above 5' protecting group does not need to be a phosphate.

[0085] The above-mentioned RNA ligases include, but are not limited to, ligase-1 (SEQ ID NO: 1), ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-5 (SEQ ID NO: 5), ligase-6 (SEQ ID NO: 6), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-12 (SEQ ID NO: 12), ligase-13 (SEQ ID NO: 13), ligase-14 (SEQ ID NO: 14), ligase-15 (SEQ ID NO: 15), ligase-16 (SEQ ID NO: 16), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-2 (SEQ ID NO: 20) or more.

[0086] In a preferred embodiment, when there are two substrates and the substrate is double-stranded RNA, the substrate includes a sense strand and an antisense strand, and the substrate includes a first substrate and a second substrate; the 3' end of the sense strand of the first substrate is a 3' hydroxyl group, and the 5' end of the antisense strand of the first substrate is a 5' phosphate group; the 5' end of the sense strand of the second substrate is a 5' phosphate group, and the 3' end of the antisense strand of the second substrate is a 3' hydroxyl group; at least one of the 5' phosphate groups of the antisense strand of the first substrate and the sense strand of the second substrate is a 5' thiophosphate; the 3' end of the sense strand of the first substrate and the 5' end of the sense strand of the second substrate are ligated under the catalysis of RNA ligase; the 5' end of the antisense strand of the first substrate and the 3' end of the antisense strand of the second substrate are ligated under the catalysis of RNA ligase; Preferably, the 3′ end of the antisense strand of the first substrate and the 3′ end of the sense strand of the second substrate are both 3′ protecting groups; preferably, the 5′ end of the sense strand of the first substrate and the 5′ end of the antisense strand of the second substrate are both 5′ protecting groups; preferably, the first substrate and the second substrate are both double-stranded RNAs with sense strands and antisense strands of different lengths, and the 3′ end of the sense strand of the first substrate is complementary to the 3′ end of the antisense strand of the second substrate, or the 5′ end of the antisense strand of the first substrate is complementary to the 5′ end of the sense strand of the second substrate, so that the 3′ end of the sense strand of the first substrate is adjacent to the 5′ end of the sense strand of the second substrate, and the 5′ end of the antisense strand of the first substrate is adjacent to the 3′ end of the antisense strand of the second substrate.

[0087] Using the above preparation method, two double-stranded RNAs can be linked via two phosphodiester bonds, at least one of which is a phosphorothioate bond, to obtain a linear double-stranded RNA. Preferably, to prevent the substrate from cyclizing itself, the corresponding 3' end of the single strand containing a 5' phosphate is not a hydroxyl group. When the first and second substrates are both double-stranded RNAs with different lengths of sense and antisense strands, and some bases in the first substrate can complementarily pair with some bases in the second substrate, it means that there are sticky ends in the first and second substrates that can complement each other. Using such substrates for ligation can achieve better ligation efficiency.

[0088] The above-mentioned RNA ligase includes, but is not limited to, any one or more of ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-5 (SEQ ID NO: 5), ligase-6 (SEQ ID NO: 6), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-14 (SEQ ID NO: 14), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-20 (SEQ ID NO: 20), and ligase-22 (SEQ ID NO: 22).

[0089] The above-mentioned 5' protecting groups include, but are not limited to, hydroxyl groups, N-acetylgalactosamine groups, phosphate groups, -O-NH2, azido groups, cyanoethyl groups, allyl groups, or 2-nitrobenzyl groups. The above-mentioned 3' protecting groups include, but are not limited to, phosphate groups, N-acetylgalactosamine groups, -O-NH2, azido groups, cyanoethyl groups, allyl groups, or 2-nitrobenzyl groups. The 3' end of the antisense strand of the first substrate and the 3' end of the sense strand of the second substrate are each independently selected from the above-mentioned 3' protecting groups, and may not be hydroxyl groups. The 5' end of the sense strand of the first substrate and the 5' end of the antisense strand of the second substrate are each independently selected from the above-mentioned 5' protecting groups, and may not be phosphate groups.

[0090] In a preferred embodiment, the preparation method further comprises using a splint to guide the connection between the first substrate and the second substrate; preferably, the splint specifically binds to at least three bases from the 3′ end to the 5′ end of the first substrate, and the splint specifically binds to at least three bases from the 5′ end to the 3′ end of the second substrate; preferably, the base on the splint that specifically binds to the 3′ end of the first substrate and the base that specifically binds to the 5′ end of the second substrate are adjacent.

[0091] In the above preparation method, a splint can be used to guide the ligation of the first and second substrates. Both the first and second substrates can specifically bind to the splint. After binding, a nick is formed between the 3' end of the first substrate and the 5' end of the second substrate. The RNA ligase can connect the nick via a phosphorothioate bond, thereby achieving the preparation of RNA containing phosphorothioate bonds. The splint comprises a DNA sequence or an RNA sequence.

[0092] The above-mentioned RNA ligase includes, but is not limited to, any one or more of ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-5 (SEQ ID NO: 5), ligase-6 (SEQ ID NO: 6), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-14 (SEQ ID NO: 14), ligase-16 (SEQ ID NO: 16), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-20 (SEQ ID NO: 20), ligase-21 (SEQ ID NO: 21), and ligase-22 (SEQ ID NO: 22).

[0093] In a preferred embodiment, the preparation method is repeatedly utilized to connect three or more substrates multiple times, with two substrates connected each time, and RNA containing phosphorothioate bonds is obtained by sequential connection.

[0094] In a preferred embodiment, the preparation method includes using a splint to guide the connection of three or more substrates; preferably, the splint and the substrate both have at least three base-specific bindings; preferably, the bases of the substrate specifically bound to the splint are adjacent, and the adjacent bases are the 5' end and 3' end of different substrates, respectively, the 5' end is a 5' phosphate, at least one of the 5' phosphates is a 5' thiophosphate, and more preferably all are 5' thiophosphates; and the 3' end is a hydroxyl group.

[0095] Using the above preparation method, multiple substrates can be connected simultaneously using a splint. The above RNA ligase can connect the nicks between multiple substrates to obtain RNA containing one or more phosphorothioate bonds.

[0096] Figure 1 is a schematic diagram of the above-mentioned preparation method, wherein Figure 1a shows a schematic diagram of connecting two single-stranded RNAs using the above-mentioned method; Figure 1b shows a schematic diagram of using a splint to guide the connection of two single-stranded RNA substrates using the above-mentioned preparation method; Figure 1c shows a schematic diagram of using the above-mentioned preparation method to connect two double-stranded RNAs; and Figure 1d shows a schematic diagram of using the above-mentioned preparation method to cyclize the substrates to form single-stranded circular RNA. As shown in Figure 1d, for the cyclization of single-stranded RNA, there are two methods, including using a splint to guide the cyclization of single-stranded circular RNA, and using RNA ligase to directly catalyze the cyclization of single-stranded RNA, which is not conducive to the cyclization of single-stranded RNA.

[0097] In a preferred embodiment, the catalytic system of RNA ligase comprises: 1-20000 μM of the substrate, 1-40000 μM ATP, 5-100 mM Tris-HCl, 0.1-20 mM divalent metal ions, 0-10 mM dithiothreitol, and 0.001-10 mg / mL RNA ligase; preferably, the temperature for RNA ligase catalysis is 4-50°C, including but not limited to 4, 10, 15, 20, 25, 30, 35, 40, 45 or 50°C; the time for RNA ligase catalysis is 0.1-24h, including but not limited to 0.1, 0.5, 1, 2, 3, 4, 5, 7, 10, 15, 20 or 24h; preferably, the divalent metal ions are including but not limited to magnesium ions, and the divalent metal ions are derived from reagents including but not limited to MgCl2 or MgSO4.

[0098] The concentration of the substrate includes, but is not limited to, 1, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 8000, 10000, 15000, or 20000 μM; the concentration of ATP includes, but is not limited to, 1, 10, 20, 50, 100, 200, 500, 1000, 1500, 2000, 3000, 4000, 5000, 8000, 10000, 20000, or 40000 μM; the concentration of RNA ligase includes, but is not limited to, 0.001, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2, 5, or 10 mg / mL.

[0099] In a preferred embodiment, the substrate includes one or more non-natural nucleotides, and the RNA containing a phosphorothioate bond includes a non-natural RNA containing a phosphorothioate bond; preferably, the number of non-natural nucleotides in the substrate is ≥2; preferably, the 5' end and / or 3' end of the substrate is a non-natural nucleotide; preferably, the substrate is composed of non-natural nucleotides; preferably, the non-natural nucleotides include: ribonucleotides with one or more of the following modifications: ribose 2' position modification, ribose backbone modification, base modification or phosphate backbone modification; preferably, the ribose 2' position modification includes 2'-methoxy modification, 2'-fluorine modification, 2'-hydrogen modification, 2'-methoxyethyl modification, 2'-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; preferably, the ribose backbone modification includes replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA or dXNA; preferably, the base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; the phosphate backbone modification includes PS modification.

[0100] Among them, the locked nucleic acid is modified to Hexitol nucleic acid modification 2′-methoxyethyl modified 2′ methoxy modified 2′ fluorine modified 2′-FANA ribuloNA is TNA tPhoNA dXNA is PS modified to In the above structure, Base represents a base. For the above modification of non-natural nucleotides, please refer to the literature: Duffy K, Arangundy-Franklin S, Holliger P. Modified nucleic acids: replication, evolution, and next-generation therapeutics [J]. BMC Biology, 2020, 18 (1): 112. Non-natural nucleotides (xeno-nucleic acid, XNA) are a class of nucleic acid molecules with non-natural backbones or nucleic acid bases. Non-natural ribonucleotides are ribonucleotides with non-natural backbones or nucleic acid bases. The above substrate contains non-natural nucleotides, and the RNA prepared by ligation contains non-natural nucleotides, which belong to non-natural tRNA. If the 3′-end base of the substrate donor and / or the 5′-end base of the substrate acceptor is a non-natural base, although the base at the junction is different from the natural base and is an artificially created non-natural base, the above RNA ligase can also exert ligation activity.

[0101] In non-natural nucleotides, the 2′ modification increases the steric hindrance of the 2′ group, such as the methoxy group, or changes the properties of the 2′ group, such as the fluorinated group. The preparation method disclosed in the present application can connect 2′-modified nucleic acids, so the above preparation method can also be used to connect natural nucleic acids that serve as natural substrates of ligase and have a 2′ hydroxyl group.

[0102] In a second typical embodiment of the present application, an RNA containing a phosphorothioate bond is provided. The RNA containing a phosphorothioate bond includes the RNA containing a phosphorothioate bond prepared by the above-mentioned preparation method.

[0103] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0104] Example 1: Ligation of single-stranded RNA containing thiolation modification:

[0105] In this example, two single-stranded non-natural RNA substrates, F1 and sF2, were designed. F1 is 13 nt long, with a 5′ hydroxyl group and a 3′ hydroxyl group; sF2 is 10 nt long, with a 5′ phosphothioate modification and a 3′ protecting group blocking its 3′ end.

[0106] F1: SEQ ID NO: 23: UmCmCmUmUmAmGfUmCfAfGfUmAm.

[0107] sF2: SEQ ID NO: 24: GmAmUmAmUmUmUmAmUm.

[0108] The m after A, C, G or U represents a 2'-methoxy modification of the ribonucleotide, and the f represents a 2'-fluoro modification of the ribonucleotide.

[0109] F1 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8, 12, and 13, and 2' fluorine modifications on ribonucleotides at positions 7, 9, 10, and 11.

[0110] sF2 has 2' methoxy modifications on ribonucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0111] The 3' hydroxyl group of F1 and the 5' thiophosphate of sF2 form a thiophosphate bond under the catalysis of RNA ligase, connecting into a 23nt single-stranded RNA.

[0112] RNA ligase was used for the ligation reaction. The ligation reaction system was prepared as 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT (dithiothreitol), 400 μM ATP, 200 μM F1, and 200 μM sF2. The reaction was carried out at 16°C for 16 hours. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 minutes.

[0113] The reaction system was detected by urea denaturing polyacrylamide gel electrophoresis. The gel electrophoresis detection results are shown in Figure 2. Blank is a negative control group without adding RNA ligase. Compared with blank, RNA ligase ligase-1 (SEQ ID NO: 1), ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-5 (SEQ ID NO: 5), ligase-6 (SEQ ID NO: 6), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-12 (SEQ ID NO: 12), ligase-13 (SEQ ID NO: 13), ligase-14 (SEQ ID NO: 14), ligase-15 (SEQ ID NO: 15), ligase-16 (SEQ ID NO: 16), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-20 (SEQ ID NO: 21), ligase-21 (SEQ ID NO: 22), ligase-22 (SEQ ID NO: 23), ligase-23 (SEQ ID NO: 24), ligase-24 (SEQ ID NO: 25), ligase-25 (SEQ ID NO: 26), ligase-26 (SEQ ID NO: 27), ligase-27 (SEQ ID NO: A new RNA band was detected between 21 nt and 25 nt in the reaction system of ligase-1 (SEQ ID NO: 16), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), and ligase-20 (SEQ ID NO: 20). The molecular weight of the new band detected by liquid chromatography-mass spectrometry was consistent with the theoretical molecular weight of the F1 and sF2 ligation product.

[0114] In the RNA ligase ligase-23 (Methanothermobacter thermautotrophicus, RNA ligase 3, NCBI Reference Sequence: WP_048060995.1), ligase-24 (Naegleria gruberi, RNA ligase 5, NCBI Reference Sequence: XP_002669268.1), ligase-25 (Pyrococcus horikoshii, RNA ligase RtcB, PDB: 4DWR_A), ligase-26 (Thermococcus barophilus, RNA ligase 1, NCBI Reference Sequence: WP_013466453.1), ligase-27 (Cronobacter phage, RNA ligase 2, NCBI Reference No ligation product band was detected in the reaction system of RNA ligase (Sequence: YP_003858534.1), indicating that only a specific wild-type RNA ligase can catalyze the ligation reaction of single-stranded RNA containing thiolation modification.

[0115] The catalytic activity of RNA ligase was analyzed by UPLC as shown in Table 1.

[0116] Table 1

[0117] Note: "+" indicates a product system purity of 0-1% (excluding the endpoint of 0%), "++" indicates a product system purity of 1-2% (excluding the endpoint of 1%), "+++" indicates a product system purity of 2-3% (excluding the endpoint of 2%), and "++++" indicates a product system purity of 3-4% (excluding the endpoint of 3%). In this application, "product system purity" refers to the ratio of the target product in the reaction product to the total components in the reaction system (target product + remaining substrate), which can be calculated by calculating the area under the curve of different components in the liquid chromatography results.

[0118] Example 2: Ligation of single-stranded RNA containing thiolation modification (splint method):

[0119] In this example, two single-stranded non-natural RNA substrates, P11 and P15, and a splint, P14, were designed. P11 is 9 nt long with a hydroxyl group at the 3' end; P15 is 6 nt long with a phosphothioate modification at the 5' end; P14 is a 15 nt RNA sequence; and P13 is a 15 nt single-stranded RNA without a phosphothioate modification that complements P14.

[0120] P11:AmCfGmGfGmGfUmCfAm.

[0121] P15: CfUmGfAmGfUm.

[0122] P14: SEQ ID NO: 25: AmCfUmCfAmGfUmGfAmCfCmCfCmGfUm.

[0123] P13: SEQ ID NO: 26: AmCfGmGfGmGfUmCfAmCfUmGfAmGfUm.

[0124] The m after A, C, G or U represents a 2'-methoxy modification of the ribonucleotide, and the f represents a 2'-fluoro modification of the ribonucleotide.

[0125] P11 has 2' methoxy modifications on ribonucleotides 1, 3, 5, 7, and 9, and 2' fluorine modifications on ribonucleotides 2, 4, 6, and 8.

[0126] P15 has 2' methoxy modifications on ribonucleotides at positions 2, 4, and 6, and 2' fluorine modifications on ribonucleotides at positions 1, 3, and 5.

[0127] P14 has 2' methoxy modifications on ribonucleotides 1, 3, 5, 7, 9, 11, 13, and 15, and 2' fluorine modifications on ribonucleotides 2, 4, 6, 8, 10, 12, and 14.

[0128] P13 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, and 15, and 2' fluorine modifications on ribonucleotides at positions 2, 4, 6, 8, 10, 12, and 14.

[0129] P11 and P15 anneal with the splint P14 to form a double-stranded structure with a gap and complementary pairing. Under the catalysis of RNA ligase, the 3' hydroxyl group of P11 and the 5' thiophosphate of P15 form a phosphorothioate bond, connecting into a 15nt RNA.

[0130] RNA ligase was used for ligation reaction. The ligation reaction system was prepared as 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 20 μM P11, 20 μM P15, and 20 μM P14. The reaction was carried out at 16°C for 16 hours. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 minutes.

[0131] The reaction system was detected by urea denaturing polyacrylamide gel electrophoresis. The gel electrophoresis detection results are shown in Figure 3. Blank is a negative control group without adding RNA ligase. Compared with blank, RNA ligase ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-6 (SEQ ID NO: 6), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-14 (SEQ ID NO: 14), ligase-16 (SEQ ID NO: 16), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-20 (SEQ ID NO: 20), ligase-21 (SEQ ID NO: 21), ligase-22 (SEQ ID NO: 22), ligase-23 (SEQ ID NO: 23), ligase-24 (SEQ ID NO: 24), ligase-25 (SEQ ID NO: 25), ligase-26 (SEQ ID NO: 26), ligase-27 (SEQ ID NO: 27), ligase-28 (SEQ ID NO: 28), ligase-29 (SEQ ID NO: 29), ligase-30 (SEQ ID NO: 30), ligase-31 (SEQ ID NO: 31), ligase-32 (SEQ ID NO: 32), ligase-33 (SEQ ID NO: 33), ligase-34 (SEQ ID NO: A new RNA band was detected in the reaction system of the P13+P14 standard product (SEQ ID NO: 20), ligase-21 (SEQ ID NO: 21), and ligase-22 (SEQ ID NO: 22). Mass spectrometry analysis detected the presence of a new molecular weight in the reaction system that was consistent with the theoretical molecular weight of the P11 and P15 ligation product.

[0132] In the RNA ligase ligase-23 (Methanothermobacter thermautotrophicus, RNA ligase 3, NCBI Reference Sequence: WP_048060995.1), ligase-24 (Naegleria gruberi, RNA ligase 5, NCBI Reference Sequence: XP_002669268.1), ligase-25 (Pyrococcus horikoshii, RNA ligase RtcB, PDB: 4DWR_A), ligase-26 (Thermococcus barophilus, RNA ligase 1, NCBI Reference Sequence: WP_013466453.1), ligase-27 (Cronobacterphage, RNA ligase 2, NCBI Reference No ligation product band was detected in the reaction system of RNA ligase (Sequence: YP_003858534.1), indicating that only a specific wild-type RNA ligase can catalyze the splint ligation reaction of single-stranded RNA containing thiolation modification.

[0133] The UPLC spectrum before the reaction is shown in Figure 4, and the spectrum after the reaction is shown in Figure 5. Comparison shows that the complementary pairing of splint P14 and the ligation product forms a new reaction product peak. UPLC analysis of the catalytic activity of RNA ligase is shown in Table 2.

[0134] Table 2

[0135] Note: “+” indicates a product system purity of 0-10% (excluding the endpoint value of 0%), “++” indicates a product system purity of 10-20% (excluding the endpoint value of 10%), “+++” indicates a product system purity of 20-30% (excluding the endpoint value of 20%), and “++++” indicates a product system purity of 30-40% (excluding the endpoint value of 30%).

[0136] Example 3: Ligation of single-stranded RNA containing thiolation modification (splint method):

[0137] In this example, two single-stranded non-natural RNA substrates, SN-1 and SN-2, and a DNA splint, Nus-R, were designed. SN-1 is 8 nt long, with a hydroxyl group at the 5' end and a hydroxyl group at the 3' end; SN-2 is 10 nt long, with a phosphothioate modification at the 5' end and a 3' protecting group at the 3' end; and Nus-R is a 16 nt DNA sequence.

[0138] SN-1: UmsCmsAmsCmsUmsUmsUmsCms.

[0139] SN-2: SEQ ID NO: 27: AmsUmsAmsAmsUmsGmsCmsUmsGmsGms.

[0140] Nus-R: SEQ ID NO: 28: dAdGdCdAdTdTdAdTdGdAdAdAdGdTdGdA.

[0141] The m after A, C, G or U indicates a 2'-methoxy modification of the ribonucleotide, the s indicates a 5'-phosphate thio modification of the ribonucleotide, and the d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide (DNA).

[0142] SN-1 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7 and 8 and 5' phosphate thio modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7 and 8.

[0143] SN-2 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and 5' phosphate thio modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0144] The nucleotides at positions 1 to 16 of Nus-R are all deoxyribonucleotides (DNA).

[0145] The following RNA ligase was used for ligation reaction. The ligation reaction system was prepared as follows: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 100 μM SN-1, 100 μM SN-2, 100 μM Nus-R, 0.2 mg / mL ligase, and the reaction was carried out at 16°C for 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min.

[0146] The UPLC detection results are shown in Figure 6. A new peak was detected in the reaction system of RNA ligases ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-5 (SEQ ID NO: 5), ligase-6 (SEQ ID NO: 6), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-14 (SEQ ID NO: 14), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-20 (SEQ ID NO: 20), and ligase-22 (SEQ ID NO: 22). The new peak was identified by liquid chromatography-mass spectrometry, and the results are shown in Figure 7. The molecular weight of the detected new peak is consistent with the theoretical molecular weight of the SN-1 and SN-2 ligation products.

[0147] Example 4: Ligation of single-stranded RNA containing thiolation modification:

[0148] In this example, two single-stranded non-natural RNA substrates, SN-1 and SN-2, were designed. SN-1 is 8 nt long, with a 5′ hydroxyl group and a 3′ hydroxyl group; SN-2 is 10 nt long, with a 5′ phosphothioate modification and a 3′ protecting group.

[0149] SN-1: UmsCmsAmsCmsUmsUmsUmsCms.

[0150] SN-2: SEQ ID NO: 27: AmsUmsAmsAmsUmsGmsCmsUmsGmsGms.

[0151] The m after A, C, G or U represents a 2′-methoxy modification of the ribonucleotide, and the s represents a thio modification of the 5′-phosphate of the ribonucleotide.

[0152] SN-1 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7 and 8 and 5' phosphate thio modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7 and 8.

[0153] SN-2 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and 5' phosphate thio modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0154] RNA ligase was used for ligation reaction. The ligation reaction system was prepared as 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 100 μM SN-1, 100 μM SN-2, and 0.2 mg / mL ligase. The reaction was carried out at 16°C for 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min.

[0155] The UPLC detection results are shown in Figure 8 . A new peak was detected in the reaction system of RNA ligases ligase-1 (SEQ ID NO: 1), ligase-4 (SEQ ID NO: 4), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-12 (SEQ ID NO: 12), ligase-13 (SEQ ID NO: 13), ligase-14 (SEQ ID NO: 14), ligase-15 (SEQ ID NO: 15), ligase-16 (SEQ ID NO: 16), ligase-18 (SEQ ID NO: 18), and ligase-19 (SEQ ID NO: 19). The new peak was identified by liquid chromatography-mass spectrometry, and the results are shown in Figure 9 . The molecular weight of the detected new peak is consistent with the theoretical molecular weight of the SN-1 and SN-2 ligation product.

[0156] Example 5: Ligation of double-stranded RNA containing thiolation modification:

[0157] In this example, four single-stranded non-natural RNAs were designed: d1 is 12 nt long, with a hydroxyl group at the 5' end and a hydroxyl group at the 3' end; d2 is 9 nt long, with a phosphothioate modification at the 5' end and a 3' protecting group at the 3' end; d3 is 9 nt long, with a phosphothioate modification at the 5' end; and d4 is 14 nt long, with a hydroxyl group at the 3' end. D1 and d3 can complement each other to form double-stranded RNAs, and d2 and d4 can complement each other to form double-stranded RNAs. These two double-stranded RNAs can serve as two substrates for double-stranded RNA ligation.

[0158] d1: SEQ ID NO: 29: UmAmsCmsUmCmUmGfAmGfUfCfUm.

[0159] d2:AmsUmUmGmAmUmAmUmsUms.

[0160] d3: CmsAfGmAmGmUmAmUmsAms.

[0161] d4: SEQ ID NO: 30: AmAfsUmsAmUmCfAmAmUmAmGmAmCmUf.

[0162] The m after A, C, G or U represents a 2′-methoxy modification of the ribonucleotide, the f represents a 2′-fluoro modification of the ribonucleotide, and the s represents a 5′-thio modification of the phosphate group of the ribonucleotide.

[0163] The ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8, and 12 of d1 have 2' methoxy modifications, the ribonucleotides at positions 7, 9, 10, and 11 have 2' fluoro modifications, and the ribonucleotides at positions 2 and 3 have 5' phosphate thio modifications.

[0164] The ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, and 9 of d2 have 2' methoxy modifications, and the ribonucleotides at positions 1, 8, and 9 have 5' phosphate thio modifications.

[0165] The ribonucleotides at positions 1, 3, 4, 5, 6, 7, 8, and 9 of d3 have 2' methoxy modifications, the ribonucleotide at position 2 has a 2' fluoro modification, and the ribonucleotides at positions 1, 8, and 9 have 5' phosphate thio modifications.

[0166] The ribonucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, and 13 of d4 have 2' methoxy modifications, the ribonucleotides at positions 2, 6, and 14 have 2' fluoro modifications, and the ribonucleotides at positions 2 and 3 have 5' phosphate thio modifications.

[0167] RNA ligase was used for ligation reaction. The ligation reaction system was prepared as 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 50 μM d1, 50 μM d2, 50 μM d3, 50 μM d4, and 0.2 mg / mL ligase. The reaction was carried out at 16°C for 16 h. After the reaction was completed, the reaction was terminated by incubation at 80°C for 5 min.

[0168] The UPLC detection results are shown in Figure 10. Two new peaks corresponding to the product standards were detected in the reaction system of RNA ligase ligase-2 (SEQ ID NO: 2), ligase-3 (SEQ ID NO: 3), ligase-4 (SEQ ID NO: 4), ligase-5 (SEQ ID NO: 5), ligase-6 (SEQ ID NO: 6), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-14 (SEQ ID NO: 14), ligase-17 (SEQ ID NO: 17), ligase-18 (SEQ ID NO: 18), ligase-19 (SEQ ID NO: 19), ligase-20 (SEQ ID NO: 20), and ligase-22 (SEQ ID NO: 22). Mass spectrometry identification of the two product peaks was consistent with the theoretical molecular weight of the ligation product.

[0169] Example 6 Cyclization of single-stranded RNA containing thiolation modification:

[0170] In this example, a single-stranded non-natural RNA substrate C1 was designed, which was 24 nt in length, 5′ phosphothioate modified, and 3′ hydroxyl group.

[0171] C1: SEQ ID NO: 31: GmsAmCmUmGmAmCmAmGmAmUmGmGmCmCmUmAmUmAmAmCmGmUmGm.

[0172] The number following A, C, G or U indicates a 2′-methoxy modification of the ribonucleotide, and the number following s indicates a 5′-thio modification of the phosphate group of the ribonucleotide.

[0173] C1 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, and a 5' phosphate thio modification on ribonucleotide position 1.

[0174] RNA ligase was used for cyclization reaction. The ligation reaction system was prepared as 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 20 μM ATP, 10 μM C1, and 0.2 mg / mL ligase, and the reaction was carried out at 37°C for 16 h.

[0175] The molecular weights of the products were detected by mass spectrometry analysis in the reaction systems of RNA ligase ligase-1 (SEQ ID NO: 1), ligase-4 (SEQ ID NO: 4), ligase-7 (SEQ ID NO: 7), ligase-8 (SEQ ID NO: 8), ligase-9 (SEQ ID NO: 9), ligase-10 (SEQ ID NO: 10), ligase-11 (SEQ ID NO: 11), ligase-12 (SEQ ID NO: 12), ligase-13 (SEQ ID NO: 13), ligase-14 (SEQ ID NO: 14), ligase-15 (SEQ ID NO: 15), ligase-16 (SEQ ID NO: 16), ligase-18 (SEQ ID NO: 18), and ligase-19 (SEQ ID NO: 19). The bands in the reaction systems were analyzed by urea-denaturing polyacrylamide gel electrophoresis. As shown in Figure 11, a new band was detected below the C1 band, which is the cyclization product band.

[0176] Currently, the primary method for synthesizing RNA is solid-phase synthesis. The target RNA requires multiple accumulations. Furthermore, as the RNA chain length increases, the yield decreases and the resulting product contains more impurities, leading to a cumbersome purification process and significantly increased costs. To address the shortcomings of solid-phase synthesis, many novel methods for synthesizing RNA are under investigation, including the use of RNA ligases for RNA synthesis. RNA ligases are enzymes that can connect short RNA fragments, offering advantages such as high efficiency, low cost, and environmental friendliness.

[0177] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: In this application, a method for preparing RNA containing a phosphorothioate bond is developed. By using the above preparation method and RNA ligase, the 3' hydroxyl group of the substrate is connected to the 5' thiophosphate, thereby preparing RNA containing a phosphorothioate bond that is difficult to obtain in the prior art.

[0178] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing RNA containing phosphorothioate bonds, characterized in that: The preparation method comprises: Using RNA ligase to catalyze the connection between the 3′ hydroxyl group of the substrate and the 5′ thiophosphate to form the RNA containing the thiophosphate bond; The 5′ thiophosphate includes a 5′ phosphate containing a thio modification, wherein the thio modification includes that the oxygen atom in P-OH or P═O of the 5′ phosphate is replaced by a sulfur atom, and correspondingly becomes P-SH or P═S; The RNA ligase includes any one or more enzymes of RNA ligase family 1, 2 or 3.

2. The preparation method according to claim 1, characterized in that: The RNA ligase comprises one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22; or An enzyme having 70% or more identity with any one of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 22, and a protein having the activity of catalyzing the ligation of a 3' hydroxyl group to a 5' thiophosphate.

3. The preparation method according to claim 1, characterized in that: The substrate includes single-stranded RNA or double-stranded RNA, and correspondingly, the RNA containing phosphorothioate bonds includes single-stranded RNA containing phosphorothioate bonds or double-stranded RNA containing phosphorothioate bonds; The substrates include 1, 2 or more.

4. The preparation method according to claim 3, characterized in that: When the substrate is one and the substrate is the single-stranded RNA, the 5′ end of the substrate is the 5′ thiophosphate, and the 3′ end of the substrate is the 3′ hydroxyl group. The 5' end of the substrate and the 3' end of the substrate are connected and cyclized under the catalysis of the RNA ligase to obtain the circular single-stranded RNA containing phosphorothioate bonds.

5. The preparation method according to claim 4, characterized in that: A splint is used to guide the circularization of the single-stranded RNA, wherein the splint specifically binds to at least three bases from the 3′ end to the 5′ end of the single-stranded RNA, and the splint specifically binds to at least three bases from the 5′ end to the 3′ end of the single-stranded RNA, and the base on the splint that specifically binds to the 3′ end of the single-stranded RNA is adjacent to the base that specifically binds to the 5′ end of the single-stranded RNA.

6. The preparation method according to claim 3, characterized in that: When there are two substrates, and the substrate is the single-stranded RNA, the substrate includes a first substrate and a second substrate, the 3' end of the first substrate is the 3' hydroxyl group, and the 5' end of the second substrate is the 5' thiophosphate, The 3' end of the first substrate and the 5' end of the second substrate are connected under the catalysis of the RNA ligase to obtain the linear single-stranded RNA containing phosphorothioate bonds.

7. The preparation method according to claim 6, characterized in that: The 3' end of the second substrate is a 3' protecting group, and the 5' end of the first substrate is a 5' protecting group.

8. The preparation method according to claim 3, characterized in that: When there are two substrates, and the substrate is the double-stranded RNA, the substrate includes a sense strand and an antisense strand, and the substrate includes a first substrate and a second substrate; The 3′ end of the sense strand of the first substrate is the 3′ hydroxyl group, and the 5′ end of the antisense strand of the first substrate is a 5′ phosphate group; The 5′ end of the sense strand of the second substrate is the 5′ phosphate, and the 3′ end of the antisense strand of the second substrate is the 3′ hydroxyl; At least one of the 5′ phosphates of the antisense strand of the first substrate and the sense strand of the second substrate is the 5′ thiophosphate; The 3′ end of the sense strand of the first substrate and the 5′ end of the sense strand of the second substrate are connected under the catalysis of the RNA ligase; the 5′ end of the antisense strand of the first substrate and the 3′ end of the antisense strand of the second substrate are connected under the catalysis of the RNA ligase; The linear double-stranded RNA containing phosphorothioate bonds is obtained.

9. The preparation method according to claim 8, characterized in that: The 3′ end of the antisense strand of the first substrate and the 3′ end of the sense strand of the second substrate are both 3′ protecting groups; The 5' end of the sense strand of the first substrate and the 5' end of the antisense strand of the second substrate are both 5' protecting groups.

10. The preparation method according to claim 9, characterized in that: The first substrate and the second substrate are both double-stranded RNAs with different lengths of sense strand and antisense strand, The 3′ end of the sense strand of the first substrate is complementary to the 3′ end of the antisense strand of the second substrate, or The 5′ end of the antisense strand of the first substrate is complementary to the 5′ end of the sense strand of the second substrate, The 3' end of the sense strand of the first substrate is adjacent to the 5' end of the sense strand of the second substrate, and the 5' end of the antisense strand of the first substrate is adjacent to the 3' end of the antisense strand of the second substrate.

11. The preparation method according to claim 6, characterized in that: The preparation method further comprises using a splint to guide the connection between the first substrate and the second substrate; The splint specifically binds to at least three bases from the 3' end to the 5' end of the first substrate, and the splint specifically binds to at least three bases from the 5' end to the 3' end of the second substrate.

12. The preparation method according to claim 11, characterized in that: The base on the splint that specifically binds to the 3' end of the first substrate is adjacent to the base that specifically binds to the 5' end of the second substrate.

13. The preparation method according to any one of claims 6 to 10, characterized in that: The preparation method is repeatedly used to connect three or more substrates for multiple times, with two substrates connected each time, and the RNA containing phosphorothioate bonds is obtained by connecting them in sequence.

14. The preparation method according to claim 3, characterized in that: The preparation method comprises using a splint to guide the connection of three or more of the substrates; The splint and the substrate both have at least three base-specific bindings.

15. The preparation method according to claim 14, characterized in that: The bases of the substrates specifically bound to the splint are adjacent, and the adjacent bases are located at the 5′ end and the 3′ end of different substrates, respectively. The 5′ end is a 5′ phosphate, and at least one of the 5′ phosphates is the 5′ thiophosphate; The 3' end is a hydroxyl group.

16. The preparation method according to claim 1, characterized in that: The catalytic system of the RNA ligase comprises: 1-20000 μM of the substrate, 1-100000 μM ATP, 5-100 mM Tris-HCl, 0.1-20 mM divalent metal ions, 0-10 mM dithiothreitol, and 0.001-10 mg / mL of the RNA ligase.

17. The preparation method according to claim 16, characterized in that: The RNA ligase catalyzes the enzyme at a temperature of 4-50°C and a catalytic time of 0.1-24h; The divalent metal ions are derived from MgCl2 or MgSO4.

18. The preparation method according to claim 1, characterized in that: The substrate comprises one or more non-natural nucleotides, and the RNA containing phosphorothioate bonds comprises non-natural RNA containing phosphorothioate bonds.

19. The preparation method according to claim 18, characterized in that: The number of the non-natural nucleotides in the substrate is ≥ 2, and the 5′ end and / or 3′ end of the substrate is the non-natural nucleotide.

20. The preparation method according to claim 19, characterized in that: The substrate consists of the non-natural nucleotide.

21. The preparation method according to any one of claims 18 to 20, characterized in that: The non-natural nucleotides include ribonucleotides with one or more of the following modifications: ribose 2′ position modification, ribose backbone modification, base modification or phosphate backbone modification; The modification of the 2′ position of ribose includes 2′-methoxy modification, 2′-fluorine modification, 2′-hydrogen modification, 2′-methoxyethyl modification, 2′-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; The ribose backbone modification includes replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA or dXNA; The base modification includes deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification or uridine C5 modification; The phosphate backbone modification includes PS modification.

22. An RNA containing a phosphorothioate bond, characterized in that: The RNA containing phosphorothioate bonds includes the RNA containing phosphorothioate bonds prepared by the preparation method according to any one of claims 1 to 21.

Images