Preparation method for non-natural RNA and product

Abstract

Provided in the present invention are a preparation method for a non-natural RNA and a product. The preparation method for the non-natural RNA comprises: connecting the 3' end of a substrate donor with the 5' end of a substrate receptor by using an RNA ligase to form a phosphodiester bond, wherein the connection does not need a template chain, and the non-natural RNA is prepared. The substrate donor and / or the substrate receptor comprise one or more non-natural nucleotides. The 5' end of the substrate receptor is a phosphoric acid group; and the 3' end of the substrate donor is a hydroxyl group. The RNA ligase comprises any one or more enzymes in RNA ligase family Rnl1 and Rnl2. The solution solves the problem in the prior art of non-natural RNA being difficult to prepare using enzyme catalysis, and the method is suitable for the field of RNA synthesis.

Description

Preparation method and product of non-natural RNA

[0001] This application is based on the Chinese application with CN application number 202311335114.7 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 synthesis, and in particular to a method for preparing non-natural RNA and a product thereof. Background Art

[0003] RNA is a carrier of genetic information found in biological cells and some viruses and viroids. For a long time, RNA was thought to be solely a molecule that transmitted information between genes and proteins. In fact, at the dawn of life, RNA was the only living molecule capable of both storing information and performing enzymatic functions. In addition to serving as a messenger for protein synthesis (mRNA), RNA also possesses crucial regulatory functions. Non-transcribed RNAs include miRNAs, siRNAs, lncRNAs, and piwiRNAs. There are over 400 miRNAs alone, regulating at least one-third of human genes. Since the 1970s, gene delivery technologies, gene cloning, and gene editing have profoundly impacted modern gene therapy. RNA editing technology has made it possible to genetically edit specific nucleotides in human cells, making it not only a research tool but also a temporary treatment for diseases caused by mutations.

[0004] In recent years, the field of RNA drugs has developed rapidly. On the one hand, small nucleic acid drugs represented by siRNA and ASO have shined in the fields of rare diseases, and on the other hand, mRNA drugs represented by mRNA vaccines have played an important role in the new crown epidemic. At present, the common synthesis method of oligonucleotides (12-30 nucleotides) is mostly solid-phase synthesis, and solid-phase synthesis is to fix nucleic acids on solid-phase carriers to complete the synthesis reaction. The most commonly used solid-phase carrier is controlled microporous glass beads (CPG), but the load capacity is limited, generally 70-80μmol / g. However, there are great disadvantages in the solid-phase synthesis of nucleotide chains. The purity of the synthesized oligonucleotide initial product is low, and the synthesis chain length is limited. It is generally used to synthesize chain lengths within 25 nucleotides. As the synthesis chain length increases, the yield decreases. When the primer is synthesized to a chain length of ~80nt, the crude product purity is only ~40%. The too low purity also affects the purification yield, resulting in a low purification yield, which greatly limits the synthesis of nucleotide chains. At the same time, the large amount of organic reagents used increases the difficulty of treating the three wastes and causes serious pollution to the environment.

[0005] Natural oligonucleotides are easily degraded in the body, have low specificity, and have toxic side effects. Therefore, pharmaceutical oligonucleotides are often chemically modified to enhance their in vivo stability, improve specificity, and reduce toxic side effects. These chemical modifications include fluorinated, methyl, and methoxyethyl modifications. Compared to traditional drugs that act at the protein level, nucleic acid drugs have significant advantages such as high specificity, high efficacy, and long-lasting efficacy. However, because natural nucleic acids are highly sensitive to endonucleases and exonucleases and are easily degraded, nucleic acid drugs use chemical modifications to improve stability, reduce immunogenicity, and enhance specificity. Nucleic acids with single chemical modifications are artificially created and not natural, so the activity of natural enzymes against non-natural nucleic acids is unknown.

[0006] Enzymatic synthesis is a green and efficient method, characterized by relatively mild reaction conditions and minimal or no organic reagent requirements, making it a promising catalytic approach. Using ligases to synthesize nucleotide chains offers green catalysis and unlimited chain length, addressing the technical bottleneck of solid-phase synthesis. However, the application of ligases to tether single-stranded RNA is largely limited to natural RNA chains, and there are few reports in the literature or patents on the enzymatic synthesis of non-natural single-stranded RNA.

[0007] Summary of the Invention

[0008] The main purpose of the present invention is to provide a method and product for preparing non-natural RNA to solve the problem in the prior art that it is difficult to prepare non-natural RNA using enzyme catalysis.

[0009] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for preparing non-natural RNA is provided, which comprises: using RNA ligase to connect the 3′ end of a substrate donor to the 5′ end of a substrate acceptor to form a phosphodiester bond, wherein the connection does not require a template chain, thereby preparing the non-natural RNA, wherein the substrate donor and / or the substrate acceptor include one or more non-natural nucleotides; the 5′ end of the substrate acceptor is a phosphate group; the 3′ end of the substrate donor is a hydroxyl group; and the RNA ligase comprises any one or more enzymes of the RNA ligase family Rnl1 and Rnl2.

[0010] 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: 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 that has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 8, SEQ ID NO: 15 to SEQ ID NO: 22 and has activity in catalyzing phosphodiester bond formation.

[0011] Furthermore, the substrate donors in the preparation method include one or n, where n is an integer greater than or equal to 2. When there are n substrate donors, the preparation method includes: connecting the first substrate donor to the first substrate acceptor to form a second substrate acceptor; connecting the second substrate donor to the second substrate acceptor to form a third substrate acceptor; until the n-1th substrate donor is connected to the n-1th substrate acceptor to form the nth substrate acceptor; and connecting the nth substrate donor to the nth substrate acceptor to form a non-natural RNA.

[0012] Furthermore, the 5' end of the first substrate acceptor is a phosphate group; the 5' end of the nth substrate donor is a 5' protecting group; the 5' end of the second substrate donor to the n-1th substrate donor is a hydroxyl group; and the 3' end of the second substrate donor to the nth substrate donor is a hydroxyl group.

[0013] Furthermore, the 5′-ends of the precursor of the second substrate receptor to the precursor of the nth substrate receptor are all 5′-protecting groups, and the 5′-protecting groups include hydroxyl groups; before connecting the second substrate receptor to the nth substrate receptor to the corresponding substrate donor, the 5′-protecting groups on the precursor of the second substrate receptor to the precursor of the nth substrate receptor are replaced with phosphate groups; using polynucleotide kinase, the 5′-ends of the precursors of the second substrate receptor to the nth substrate receptor are respectively modified with phosphate groups to obtain substrate receptors with phosphate groups at the 5′-ends, and the polynucleotide kinase is inactivated.

[0014] Furthermore, the substrate acceptor in the preparation method includes one or n, where n is an integer greater than or equal to 2; when there are n substrate acceptors, the preparation method includes: connecting a first substrate donor with a first substrate acceptor to form a second substrate donor; connecting a second substrate acceptor with a second substrate donor to form a third substrate donor; until the n-1th substrate acceptor is connected with the n-1th substrate donor to form an nth substrate donor; and connecting the nth substrate acceptor with the nth substrate donor to form a non-natural RNA.

[0015] Furthermore, the 3′ end of the substrate acceptor is a protecting group; the 3′ end of the precursor of the second substrate donor to the nth substrate donor and the precursor of the non-natural RNA are all protecting groups; before the second substrate donor to the nth substrate donor are connected to the corresponding substrate acceptor, the protecting group is deprotected to obtain a substrate donor or non-natural RNA with a hydroxyl group at the 3′ end.

[0016] Furthermore, non-natural nucleotides include: ribonucleotides having one or more of ribose 2′ position modification, ribose backbone modification, base modification or phosphate backbone modification; ribose 2′ position modification includes 2′-methoxy modification, 2′-fluoro 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.

[0017] Furthermore, the substrate donor and / or substrate acceptor contains one or more non-natural nucleotides; the 3' terminal base of the substrate donor and / or the 5' terminal base of the substrate acceptor is a non-natural nucleotide.

[0018] Furthermore, the substrate donor and / or substrate acceptor consists of a non-natural nucleotide.

[0019] Furthermore, the length of the substrate donor and the substrate acceptor are both 2-500 nt.

[0020] Furthermore, the length of the non-natural RNA is greater than 3 nt.

[0021] Furthermore, the 3' end of the substrate acceptor is a protecting group; and the 5' end of the substrate donor is a 5' protecting group.

[0022] Furthermore, the protecting group includes N-acetylgalactosamine group, phosphate group, -O-NH2, azido group, cyanoethyl group, allyl group or 2-nitrobenzyl group; the 5' protecting group includes hydroxyl group, hydrogen group or methyl group.

[0023] According to a second aspect of the present invention, a non-natural RNA is provided, which includes the non-natural RNA prepared using the above preparation method.

[0024] Applying the technical solutions of the present invention, the above-mentioned preparation method assembles non-natural RNA fragments through RNA ligase-catalyzed synthesis, thereby synthesizing longer non-natural RNA chains. This allows for specific and efficient ligation without the need for a template chain. This allows for highly efficient catalysis, significantly improving efficiency and yield compared to chemical methods for synthesizing non-natural RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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:

[0026] FIG1 shows a schematic diagram of preparing non-natural RNA by connecting multiple substrate donors and substrate acceptors according to an embodiment of the present invention.

[0027] FIG2 shows a schematic diagram of preparing non-natural RNA by connecting multiple substrate acceptors and substrate donors according to an embodiment of the present invention.

[0028] FIG3 shows the results of SDS-PAGE gel electrophoresis according to Example 2 of the present invention.

[0029] FIG4 shows the results of SDS-PAGE gel electrophoresis according to Example 3 of the present invention.

[0030] FIG5 shows a UPLC chromatogram according to Example 3 of the present invention.

[0031] FIG6 shows a UPLC chromatogram according to Example 4 of the present invention.

[0032] FIG. 7 shows a UPLC chromatogram according to Example 4 of the present invention.

[0033] FIG8 shows a schematic structural diagram of L96 according to Embodiment 4 of the present invention.

[0034] FIG9 shows a schematic structural diagram of substrates 9 , 10 and 11 according to Example 5 of the present invention. DETAILED DESCRIPTION

[0035] 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.

[0036] As mentioned in the background, the use of ligases to connect single-stranded RNA is largely limited to natural RNA chains. There are few reports in the literature or patents regarding the enzymatic synthesis of non-natural single-stranded RNA. Therefore, in this application, the inventors attempted to develop a method for preparing non-natural RNA, utilizing RNA ligase-catalyzed synthesis to assemble non-natural RNA fragments, thereby synthesizing longer non-natural RNA chains. Consequently, they proposed a series of protection schemes for this application.

[0037] In a first typical embodiment of the present application, a method for preparing non-natural RNA is provided, which comprises: using RNA ligase to connect the 3′ end of a substrate donor to the 5′ end of a substrate acceptor to form a phosphodiester bond, wherein the connection does not require a template chain, to prepare the non-natural RNA, wherein the substrate donor and / or the substrate acceptor include one or more non-natural nucleotides; the 5′ end of the substrate acceptor is a phosphate group; the 3′ end of the substrate donor is a hydroxyl group; and the RNA ligase comprises any one or more enzymes of the RNA ligase family Rnl1 and Rnl2.

[0038] In the above preparation method, RNA ligase is used to connect the substrate donor and substrate acceptor, completing the connection without the need for a template strand. The 5′ end of the substrate donor and the 3′ end of the substrate acceptor are connected via a phosphodiester bond. The 5′ end of the substrate donor is a phosphate group, and the 3′ end of the substrate acceptor is a hydroxyl group.

[0039] The number of substrate acceptors or substrate donors includes one or more. If there are multiple substrate acceptors, RNA ligase can be used to perform step-by-step ligation, where the substrate acceptor is ligated to the substrate donor to form a new substrate donor, and then the new substrate acceptor is ligated to the new substrate donor. Similarly, if there are multiple substrate donors, RNA ligase can be used to perform step-by-step ligation, where the substrate donor is ligated to the substrate acceptor to form a new substrate acceptor, and then the new substrate donor is ligated to the new substrate acceptor. The above method can achieve the connection of multiple substrate acceptors and substrate donors, realize the preparation of long-chain non-natural RNA, and prevent the influence of ligation efficiency and RNA stability due to excessively long RNA fragments.

[0040] In this application, the concepts of substrate donor and substrate acceptor do not limit the specific nature of the substrate. They only define the substrate located in the 5' direction of the RNA ligation as the substrate donor, and the substrate located in the 3' direction as the substrate acceptor. In different reactions, the same reaction substrate may be a substrate donor or a substrate acceptor.

[0041] In a preferred embodiment, 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: 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, 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 of the RNA ligases shown in SEQ ID NOs: 1 to 8, SEQ ID NOs: 15 to SEQ ID NOs: 22, and having activity of catalyzing the formation of the phosphodiester bond.

[0042] The RNA ligases described above are RNA ligases screened in this application that are capable of ligating non-natural RNAs. Non-natural RNAs include single chemically modified nucleic acids that are artificially created and not natural. Therefore, the activity of natural enzymes towards non-natural nucleic acids is unknown. In this application, through large-scale screening of existing RNA ligases, the aforementioned RNA ligases capable of ligating non-natural RNAs, independent of a template strand, were obtained.

[0043] 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.

[0044] 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).

[0045] 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).

[0046] 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:

[0047] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0048] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0049] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0050] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0051] 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.

[0052] In a preferred embodiment, the substrate donors in the preparation method include one or n, where n is an integer greater than or equal to 2. When there are n substrate donors, the preparation method includes: connecting a first substrate donor to a first substrate acceptor to form a second substrate acceptor; connecting a second substrate donor to a second substrate acceptor to form a third substrate acceptor; until the n-1th substrate donor is connected to the n-1th substrate acceptor to form the nth substrate acceptor; and connecting the nth substrate donor to the nth substrate acceptor to form the non-natural RNA.

[0053] In a preferred embodiment, the 5′ end of the first substrate acceptor is a phosphate group; the 5′ end of the nth substrate donor is a 5′ protecting group; the 5′ end of the second substrate donor to the n-1th substrate donor is a hydroxyl group; and the 3′ end of the second substrate donor to the nth substrate donor is a hydroxyl group.

[0054] In a preferred embodiment, the 5′-ends of the precursor of the second substrate receptor to the precursor of the nth substrate receptor are all 5′-protecting groups, and the 5′-protecting groups include but are not limited to hydroxyl groups; before the second substrate receptor to the nth substrate receptor are connected to the corresponding substrate donor, the 5′-protecting groups on the precursor of the second substrate receptor to the precursor of the nth substrate receptor are replaced with phosphate groups; the 5′-ends of the precursors of the second substrate receptor to the nth substrate receptor are respectively modified with phosphate groups using polynucleotide kinase to obtain substrate receptors with phosphate groups at the 5′-ends, and the polynucleotide kinase is inactivated.

[0055] The number of substrate donors is n, indicating that in the preparation method, there are n substrate donors as substrate raw materials, which are gradually connected to the substrate receptor to form a new substrate receptor; the new substrate receptor is further connected to the new substrate donor, thereby connecting the substrate receptor and n substrate donors. When using the above method to connect multiple substrate donors and substrate receptors, since the 5' end of the precursor of the newly generated substrate receptor is not a phosphate group, it is necessary to use polynucleotide kinase to modify the 5' end of the precursor of the new substrate donor formed by connecting the substrate receptor and the substrate donor (i.e., the 5' end base of the original substrate receptor) into a phosphate group, and then use RNA ligase to catalyze the connection of the hydroxyl group of the 3' end of the new substrate receptor to the phosphate group. The reaction steps are shown in Figure 1. Substrate 1 in Figure 1 is the first substrate receptor mentioned above, substrate 2 is the first substrate donor mentioned above, and substrate 3 is the second substrate donor mentioned above.

[0056] In a preferred embodiment, the substrate acceptor in the above preparation method includes one or n, where n is an integer greater than or equal to 2; when there are n substrate acceptors, the preparation method includes: connecting a first substrate donor with a first substrate acceptor to form a second substrate donor; connecting a second substrate acceptor with a second substrate donor to form a third substrate donor; until the n-1th substrate acceptor is connected with the n-1th substrate donor to form the nth substrate donor; and connecting the nth substrate acceptor with the nth substrate donor to form a non-natural RNA.

[0057] In the above preparation method, the number of substrate acceptors is n, indicating that n substrate acceptors, serving as substrate raw materials, are progressively attached to a substrate donor to form a new substrate donor; the new substrate donor is further attached to a new substrate acceptor, thereby achieving the connection between the substrate donor and n substrate acceptors. The reaction steps are shown in Figure 2. In Figure 2, substrate 1 is the first substrate donor, substrate 2 is the first substrate acceptor, and substrate 3 is the second substrate acceptor.

[0058] In a preferred embodiment, the 3′ end of the substrate acceptor is a protecting group; the 3′ end of the precursors of the second substrate donor to the nth substrate donor and the precursor of the non-natural RNA are all protecting groups; before the second substrate donor to the nth substrate donor are connected to the corresponding substrate acceptor, the protecting group is deprotected to obtain a substrate donor or non-natural RNA with a hydroxyl group at the 3′ end.

[0059] In order to prevent the substrate acceptor from self-ligating and affecting the reaction yield and product purity, the 3′ end of the substrate acceptor is a protecting group. When the substrate acceptor is connected to the substrate donor to form a new substrate donor, the 3′ end of this new substrate donor is a protecting group. The new substrate donor needs to be deprotected to obtain a new substrate donor with a hydroxyl group at the 3′ end, thereby achieving further connection with the new substrate acceptor.

[0060] In a preferred embodiment, non-natural nucleotides include but are not limited to: ribonucleotides having one or more of ribose 2′ position modification, ribose backbone modification, base modification or phosphate backbone modification; preferably, ribose 2′ position modification includes but is not limited to 2′-methoxy modification, 2′-fluoro modification, 2′-methoxyethyl modification, 2′-FANA modification, locked nucleic acid modification or hexitol nucleic acid modification; preferably, ribose backbone modification includes but is not limited to replacing the ribose in the nucleotide with ribuloNA, TNA, tPhoNA, or dXNA; preferably, base modification includes but is not limited to deazaadenine C7 modification, deazaguanosine C7 modification, cytosine C5 modification, or uridine C5 modification; phosphate backbone modification includes PS modification.

[0061] 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 structures, Base represents a base, and R represents a hydroxyl, hydrogen, methoxy, methoxyethyl, or fluorine modification. For the above modifications 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.

[0062] In a preferred embodiment, the substrate donor and / or substrate acceptor contains one or more non-natural nucleotides; preferably, the 3′-terminal base of the substrate donor and / or the 5′-terminal base of the substrate acceptor is a non-natural nucleotide; preferably, the substrate donor and / or substrate acceptor consists of non-natural nucleotides.

[0063] Non-natural nucleotides (xeno-nucleic acids, 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. If the substrate donor and / or substrate acceptor contain non-natural nucleotides, the RNA prepared by ligation contains non-natural nucleotides and is non-natural RNA. If the 3′-terminal base of the substrate donor and / or the 5′-terminal base of the substrate acceptor is a non-natural nucleotide, the RNA ligase described above can still exert ligation activity, even though the bases at the ligation site are different from natural bases and are artificially created non-natural bases.

[0064] In a preferred embodiment, the length of the substrate donor and the substrate acceptor are both 2-500 nt, including but not limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400 and 500 nt.

[0065] In a preferred embodiment, the length of the non-natural RNA is greater than 3 nt, and the non-natural RNA is formed by connecting a substrate donor and a substrate acceptor having the above length, and by connecting one or more substrate acceptors and substrate donors, a non-natural RNA with a length greater than 3 nt can be obtained. The length of the non-natural RNA includes but is not limited to 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nt.

[0066] In a preferred embodiment, the 3' end of the substrate acceptor is a protecting group; preferably, the 5' end of the substrate donor is a 5' protecting group; preferably, the protecting group includes GalNac (N-acetylgalactosamine group), phosphate group, -O-NH2, azido group (-CN=N + =N - , 3′-O-azidomethyl), cyanoethyl (-C-CN, 3′-O-2-cyanoethyl), allyl (-CC=C, 3′-O-allyl), 2-nitrobenzyl (3′-O-2-nitrobenzyl); preferably, the 5′ protecting group includes hydroxyl, hydrogen or methyl.

[0067] In a second typical embodiment of the present application, a non-natural RNA is provided, which includes the non-natural RNA prepared using the above preparation method.

[0068] The sequences, sources and families of the ligases used in this application are as follows.

[0069] Ligase 1, SEQ ID NO: 1, Bacteriophage, RNA ligase 2:

[0070] Ligase 2, SEQ ID NO: 2, Vibrio phage, RNA ligase 2:

[0071] Ligase 3, SEQ ID NO: 3, Bacteriophage AR1, RNA ligase 1:

[0072] Ligase 4, SEQ ID NO: 4, Bacteriophage dhaeg, RNA ligase 2:

[0073] Ligase 5, SEQ ID NO: 5, Klebsiella phage KP179, RNA ligase 1:

[0074] Ligase 6, SEQ ID NO: 6, Vibrio phage, RNA ligase 1:

[0075] Ligase 7, SEQ ID NO: 7, Escherichia phage JN02, RNA ligase 1:

[0076] Ligase 8, SEQ ID NO: 8, Klebsiella phage KP15, RNA ligase 2:

[0077] Ligase 9, SEQ ID NO: 9, Thermococcus, RNA ligase 2-like:

[0078] Ligase 10, SEQ ID NO: 10, Archaea, tRNA-splicing ligase:

[0079] Ligase 11, SEQ ID NO: 11, thermophilic bacteriophage, RNA ligase 1:

[0080] Ligase 12, SEQ ID NO: 12, Diplonema papillatum, RNA ligase 2:

[0081] Ligase 13, SEQ ID NO: 13, virus, RNA ligase 2-like:

[0082] Ligase 14, SEQ ID NO: 14, Archaea, RNA ligase 3:

[0083] Ligase 15, SEQ ID NO: 15, Trypanasoma brucei gambiense, RNA ligase 2:

[0084] Ligase 16, SEQ ID NO: 16, Acidobacteria bacterium, RNA ligase 1:

[0085] Ligase 17, SEQ ID NO: 17, Klebsiella phage KP27, RNA ligase 2:

[0086] Ligase 18, SEQ ID NO: 18, Vibrio phage, RNA ligase 2:

[0087] Ligase 19, SEQ ID NO: 19, Vibrio phage JN02, RNA ligase 2:

[0088] Ligase 20, SEQ ID NO: 20, Vibrio phage JS98, RNA ligase 2:

[0089] Ligase 21, SEQ ID NO: 21, Vibrio phage nt-1, RNA ligase 2:

[0090] Ligase 22, SEQ ID NO: 22, Vibrio phage phi-ST2, RNA ligase 2:

[0091] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0092] Example 1 Purification and preparation of RNA ligase

[0093] The gene encoding RNA ligase was constructed into the pET28a(+) vector and transformed into the Escherichia coli BL21(DE3) expression strain. The expression strain was inoculated into 5 mL of LB medium (containing 50 μg / mL) at a 0.1% inoculum and cultured at 37°C for 16 h. The resulting culture was transferred to 500 mL of LB medium at a 1% inoculum and cultured at 37°C until the OD 600 =0.6, 0.1mM IPTG was added and induced at 20°C for 16h. The resulting induced culture was centrifuged to obtain bacterial sludge. The bacterial sludge was resuspended in lysis buffer (50mM Tris-Cl pH8.0, 0-500mM NaCl, 10% glycerol) to a final bacterial concentration of 10%, ultrasonically disrupted, and centrifuged to obtain a crude enzyme solution. 10mM imidazole was added to the crude enzyme solution, and the enzyme solution obtained after filtration with a 0.22μm filter membrane was purified using a nickel column. After desalting and replacing the solution, it was purified again using a strong anion column. The obtained enzyme solution was desalted and replaced with the solution and stored in 30% glycerol.

[0094] Example 2 RNA ligase catalyzes template-independent non-natural RNA ligation

[0095] Add substrate 1 and substrate 2 to a clean reagent bottle (the sequences of substrates 1 and 2 are shown in Table 1) and prepare a reaction system to a final volume of 100 μL. The concentrations of substrate 1 and substrate 2 are 500 μM, ATP is 1 mM, Tris-HCl (pH 7.5 @ 25°C) is 50 mM, MgCl2 is 10 mM, DTT (dithiothreitol) is 1 mM, and the ligase concentration is 0.2 mg / mL. The reaction system is incubated at 16°C for 16 hours. The resulting reaction system is inactivated by heating at 80°C for 5 minutes to inactivate the ligase. The precipitate is removed by centrifugation at 12,000 rpm, and the supernatant is analyzed by SDS-PAGE and UPLC. The results showed that ligase 6 and enzyme 8 could catalyze the synthesis of the final product. The SDS-PAGE electrophoresis diagram is shown in Figure 3, wherein lane 1 is a marker, lane 2 is a control group (no ligase), lane 3 is a system catalyzed by ligase 9, lane 4 is a system catalyzed by ligase 10, lane 5 is a system catalyzed by ligase 11, lane 6 is a system catalyzed by ligase 13, lane 7 is a system catalyzed by ligase 6, lane 8 is a system catalyzed by ligase 8, and lane 9 is a system catalyzed by ligase 12. The UPLC results showed that the substrate had been basically completely converted. This shows that the method of ligase-catalyzed synthesis of non-natural RNA is successful.

[0096] Table 1

[0097] Here, m after A, C, G or U represents a 2'-methoxy modification of the ribonucleotide, and f represents a 2'-fluorine modification of the ribonucleotide.

[0098] Substrate 1 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.

[0099] Substrate 2 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 5, and 6, and 2' fluorine modifications on ribonucleotides at positions 3 and 4.

[0100] However, not all ligases can achieve the splicing of non-natural RNA. Under the same reaction conditions as above, ligases 9, 10, 11, and 12 do not have this activity, and ligase 13 produces a large amount of substrate residue and the formation of impurity bands.

[0101] Example 3 RNA ligase catalyzes template-independent ligation of short non-natural RNA chains

[0102] Add substrates 3 and 4 to a clean reagent bottle (the sequences of substrates 3 and 4 are shown in Table 2) to prepare a reaction system with a final volume of 100 μL. The concentrations of RNA fragments 3 and 4 were 1000 μM, ATP was 1.5 mM, Tris-HCl (pH 7.5 @ 25°C) was 50 mM, MgCl₂ was 10 mM, DTT was 1 mM, and ligase was 0.2 mg / mL. The reaction system was incubated at 16°C for 16 h. The resulting reaction system was inactivated by heating at 80°C for 5 min to inactivate the ligase. The precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by SDS-PAGE and UPLC. The results show that in the control system without the addition of ligase (channel 3), no target product was generated, while the synthesis of the final product was found in the system with the addition of RNA ligase. The SDS-PAGE electrophoresis diagram is shown in Figure 4, where lane 1 is a system catalyzed by ligase 6, lane 2 is a system catalyzed by ligase 11, and lane 3 is a control group (no ligase). The same screening was performed for the other ligases. However, since substrates 3 and 4 are shorter, the remaining substrates cannot be clearly observed in SDS-PAGE, so UPLC was used to detect the reaction. The results show that the substrates have been basically completely converted. The UPLC chromatogram of the reaction catalyzed by ligase 7 is shown in Figure 5. This shows that the method for synthesizing non-natural RNA catalyzed by ligase is successful. The RNA ligases used and their catalytic activities are shown in Table 3.

[0103] Table 2

[0104] Here, m after A, C, G or U represents a 2'-methoxy modification of the ribonucleotide, and f represents a 2'-fluorine modification of the ribonucleotide.

[0105] Substrate 3 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 5, and 6, and 2' fluorine modifications on ribonucleotides at positions 3 and 4.

[0106] Substrate 4 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 5, 6, 9 and 10, and 2' fluorine modifications on ribonucleotides at positions 3, 4, 7 and 8.

[0107] Table 3 Note: In this application, “+++” indicates a catalytic activity of 70% to 100%, “++” indicates a catalytic activity of 60% to 70% (excluding the endpoint value of 70%), and “-” indicates substantially no catalytic activity.

[0108] Example 4 RNA ligase catalyzes template-independent ligation of long non-natural RNA chains

[0109] Substrates 7 and 8 were added to a clean reagent bottle to prepare a reaction system with a final volume of 100 μL. The concentrations of substrates 7 and 8 were 300 μM, ATP was 0.6 mM, Tris-HCl (pH 7.5 @ 25°C) was 50 mM, MgCl₂ was 10 mM, DTT was 1 mM, and ligase was 0.2 mg / mL. The reaction system was incubated at 37°C for 16 h. The resulting reaction system was inactivated by heating the ligase at 80°C for 5 min, the precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by SDS-PAGE and UPLC. The UPLC chromatogram of the reaction catalyzed by ligase 4 is shown in Figure 6. The results show the formation of product in the system with RNA ligase added, indicating that the ligase-catalyzed synthesis of non-natural RNA is successful. The RNA ligases used and their catalytic activities are shown in Table 4.

[0110] Table 4

[0111] Substrates 5 and 6 were added to a clean reagent bottle to prepare a reaction system with a final volume of 100 μL. The concentrations of substrates 5 and 6 were 100 μM, ATP was 0.2 mM, Tris-HCl (pH 7.5 @ 25°C) was 50 mM, MgCl₂ was 10 mM, DTT was 1 mM, and ligase was 0.2 mg / mL. The reaction system was incubated at 37°C for 16 h. The resulting reaction system was inactivated by heating the ligase at 80°C for 5 min, the precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by SDS-PAGE and UPLC. The UPLC chromatogram of the reaction catalyzed by ligase 5 is shown in Figure 7. The results show the formation of the final product in the system containing RNA ligase, indicating that the ligase-catalyzed synthesis of non-natural RNA is successful. The RNA ligases used and their catalytic activities are shown in Table 5.

[0112] The sequences of substrates 5, 6, 7 and 8 are shown in Table 6.

[0113] Table 5

[0114] Table 6

[0115] The structure of L96 is shown in Figure 8, where represents the base to which the L96 group is attached; m after A, C, G or U represents a 2' methoxy modification of the ribonucleotide.

[0116] All ribonucleotides in substrates 5, 6, 7, and 8 have 2' methoxy modifications.

[0117] Example 5 RNA ligase catalyzes the ligation of multiple fragments of non-natural RNA substrates

[0118] Step 1: Add substrates 10 and 11 to a clean reagent bottle and prepare a reaction system to a final volume of 100 μL. The concentrations of substrates 10 and 11 are 800 μM, ATP is 1.6 mM, Tris-HCl (pH 7.5 @ 25°C) is 50 mM, MgCl2 is 10 mM, DTT is 1 mM, and ligase is 0.2 mg / mL. Incubate the reaction system at 16°C for 16 h. Ensure that the substrates are completely consumed.

[0119] Step 2: Add 8 μL of 10 U / μL T4 polynucleotide kinase and incubate the reaction system at 37°C for 6 h. Inactivate T4 polynucleotide kinase and ligase in the resulting reaction system at 80°C for 5 min.

[0120] Step 3: Add substrate 9 in an amount equimolar to substrate 10, and add an equimolar amount of ATP to a final system substrate concentration of 200 μM. Add 0.2 mg / mL of ligase and react at 16°C for 16 hours. The resulting final reaction system inactivates the ligase at 80°C for 5 minutes, the precipitate is removed by centrifugation at 12,000 rpm, and the supernatant is detected by SDS-PAGE and UPLC. The results show that the final product is generated in the final system. The final product is composed of substrates 9, 10, and 11 connected from the 5' end to the 3' end, and the conversion rate of the product reaches over 60%, indicating that the method of RNA ligase-catalyzed synthesis of non-natural RNA is successful. The RNA ligases used and their catalytic activities are shown in Table 7.

[0121] The sequences of substrates 9, 10 and 11 are shown in Table 8, and the schematic diagrams of substrates 9, 10 and 11 are shown in Figure 9.

[0122] Table 7

[0123] Table 8

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

[0125] Substrate 9 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 5, and 6, and 2' fluorine modifications on ribonucleotides at positions 3 and 4.

[0126] Substrate 10 has 2' methoxy modifications on ribonucleotides at positions 3, 4, 7, and 8, and 2' fluorine modifications on ribonucleotides at positions 1, 2, 5, and 6.

[0127] Substrate 11 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 5, 6, 9, and 10, and 2' fluorine modifications on ribonucleotides at positions 3, 4, 7, and 8.

[0128] From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: The present invention has discovered that some RNA ligases are capable of ligating non-natural nucleotides. Utilizing the above-described preparation method and the RNA ligase-catalyzed synthesis method, two or more non-natural RNA fragments can be ligated and assembled, thereby synthesizing longer non-natural RNA chains. This allows for specific and efficient ligation without the need for a template chain. This allows for efficient catalysis, significantly improving efficiency and yield compared to chemical methods for synthesizing non-natural RNA. This also overcomes the prior art problem of difficulty in ligating non-natural nucleotides using existing RNA ligases used to ligate natural nucleic acids.

[0129] 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 non-natural RNA, characterized in that: The preparation method comprises: The 3′ end of the substrate donor and the 5′ end of the substrate acceptor are connected by RNA ligase to form a phosphodiester bond, wherein the connection does not require a template strand, thereby preparing the non-natural RNA. The substrate donor and / or the substrate acceptor comprises one or more non-natural nucleotides; The 5′ end of the substrate receptor is a phosphate group; The 3′ end of the substrate donor is a hydroxyl group; The RNA ligase includes any one or more enzymes of the RNA ligase family Rnl1 and Rnl2.

2. The preparation method according to claim 1, characterized in that: The RNA ligase comprises: one or more of the RNA ligases set forth 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: 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, and SEQ ID NO: 22; or An enzyme that has 70% or more identity with any one of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 8 and SEQ ID NO: 15 to SEQ ID NO: 22 and has an activity of catalyzing the formation of the phosphodiester bond.

3. The preparation method according to claim 1 or 2, characterized in that: The substrate donor in the preparation method includes one or n substrate donors, where n is an integer greater than or equal to 2. When the number of the substrate donors is n, the preparation method comprises: The first substrate donor is linked to the first substrate acceptor to form a second substrate acceptor; The second substrate donor is then connected to the second substrate acceptor to form a third substrate acceptor; Until the n-1th substrate donor is connected to the n-1th substrate acceptor to form the nth substrate acceptor; The nth substrate donor is linked to the nth substrate acceptor to form the non-natural RNA.

4. The preparation method according to claim 3, characterized in that: The 5′ end of the first substrate receptor is a phosphate group; The 5′ end of the nth substrate donor is a 5′ protecting group; The 5′ end of the second substrate donor to the n-1th substrate donor is a hydroxyl group; The 3′ end of the second substrate donor to the nth substrate donor is a hydroxyl group.

5. The preparation method according to claim 4, characterized in that: The 5′ ends of the precursor of the second substrate receptor to the precursor of the nth substrate receptor are all 5′ protecting groups, and the 5′ protecting groups include hydroxyl groups; Before connecting the second substrate acceptor to the nth substrate acceptor to the corresponding substrate donor, replacing the 5′ protecting group on the precursor of the second substrate acceptor to the nth substrate acceptor with a phosphate group; The 5' ends of the precursor of the second substrate receptor to the precursor of the nth substrate receptor are modified with phosphate groups respectively by using polynucleotide kinase to obtain the substrate receptor with a phosphate group at the 5' end, and the polynucleotide kinase is inactivated.

6. The preparation method according to claim 1 or 2, characterized in that: The substrate receptor in the preparation method includes one or n substrate receptors, where n is an integer greater than or equal to 2; When the number of the substrate receptors is n, the preparation method comprises: connecting a first substrate donor to the first substrate acceptor to form a second substrate donor; connecting a second substrate acceptor to the second substrate donor to form a third substrate donor; until the n-1th substrate acceptor is connected to the n-1th substrate donor to form the nth substrate donor; The nth substrate acceptor is linked to the nth substrate donor to form the non-natural RNA.

7. The preparation method according to claim 6, characterized in that: The 3′ end of the substrate acceptor is a protecting group; accordingly, the 3′ ends of the precursor of the second substrate donor obtained by connection to the precursor of the nth substrate donor are both the protecting group; Before connecting the second substrate donor to the nth substrate donor and the corresponding substrate acceptor, the protecting group is deprotected to obtain the substrate donor or the non-natural RNA with a hydroxyl group at the 3' end.

8. The preparation method according to claim 1, characterized in that: The non-natural nucleotides include: ribonucleotides with one or more of 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′-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.

9. The preparation method according to claim 1, characterized in that: The substrate donor and / or the substrate acceptor contains one or more non-natural nucleotides; The 3' terminal base of the substrate donor and / or the 5' terminal base of the substrate acceptor is the non-natural nucleotide.

10. The preparation method according to claim 9, characterized in that: The substrate donor and / or the substrate acceptor consists of the non-natural nucleotide.

11. The preparation method according to claim 1, characterized in that: The length of the substrate donor and the substrate acceptor are both 2-500 nt.

12. The preparation method according to claim 1, characterized in that: The length of the non-natural RNA is greater than 3 nt.

13. The preparation method according to claim 1, characterized in that: The 3' end of the substrate acceptor is a protecting group; the 5' end of the substrate donor is a 5' protecting group.

14. The preparation method according to claim 13, characterized in that: The protecting groups include N-acetylgalactosamine group, phosphate group, -O-NH2, azido group, cyanoethyl group, allyl group or 2-nitrobenzyl group; the 5' protecting group includes hydroxyl group, hydrogen or methyl group.

15. A non-natural RNA, characterized in that The non-natural RNA includes the non-natural RNA prepared by the preparation method described in any one of claims 1-14.

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