Trna and biosynthesis method therefor

Abstract

Provided are a tRNA and a biosynthesis method therefor. The biosynthesis method for the tRNA comprises: using an RNA ligase to connect 3' ends and 5' ends of different substrates to form phosphodiester bonds, and forming the tRNA, wherein the number of the substrates is greater than or equal to 2, and the RNA ligase comprises any one or more of RNA ligase family Rnl1, Rnl2, Rnl3 and Rnl5. The present invention can solve the problem in the prior art of low tRNA synthesis yields, and is suitable for the field of tRNA synthesis.

Description

tRNA and its biosynthesis method

[0001] This application is based on the Chinese application with CN application number 202311335092.4 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 tRNA synthesis, and in particular to a tRNA and a biosynthesis method thereof. Background Art

[0003] tRNA is a ribonucleic acid molecule consisting of 70 to 120 nucleotides in length and serves as an adaptor molecule in protein synthesis. There are over 100 different tRNA molecules, each carrying a specific amino acid, which is transported to the ribosome for use in protein synthesis. The secondary structure of tRNA is an inverted trefoil configuration, with a 3′-OH moiety at one end that binds to the amino acid and an anticodon at the other end that pairs with the codon bases on the mRNA. The RNA chain folds back on itself, and the complementary sequences pair to form a partial double helix, called the stem or arm. The unpaired bases between the two segments remain single-stranded, forming a loop. The stem and loop form a stem-loop structure, also known as a hairpin structure. Generally, the higher the proportion of RNA folding back, the more stable the structure. tRNA not only plays an important role in life, but research has also demonstrated its potential use in treating some diseases caused by gene mutations. For example, the 2021 study "RNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase" published in Science demonstrated that insufficient intracellular tRNA supply due to gene mutations contributes to the peripheral neuropathy Charcot-Marie-Tooth disease. Increasing glycyl-tRNA levels may offer potential therapeutic benefits. Therefore, tRNA therapy may become an effective treatment for human diseases in the future.

[0004] Currently, RNA synthesis is primarily achieved through solid-phase synthesis. However, the length of immobilized RNA is limited. tRNA is typically 70 to 120 nucleotides long, making it impossible to synthesize using solid-phase synthesis. Currently, research on tRNA synthesis is limited. The main synthetic pathway is derived from natural tRNA synthesis within cells, which is then extracted and obtained. This results in extremely low yields and is susceptible to nuclease contamination.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a tRNA and a biosynthesis method thereof to solve the problem of low yield in the prior art in synthesizing tRNA.

[0007] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for the biosynthesis of tRNA is provided, which comprises: using RNA ligase to connect the 3′ and 5′ ends of different substrates to form a phosphodiester bond to form tRNA, wherein the number of substrates is ≥2; the RNA ligase comprises any one or more enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3 and Rnl5.

[0008] Furthermore, the substrate includes one or / and a plurality of non-natural nucleotides, and the tRNA includes a non-natural tRNA.

[0009] 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: 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, SEQ ID NO: 22, or SEQ ID NO: 23; or a protein that has 70% or more identity with any one of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 6 or SEQ ID NO: 10 to SEQ ID NO: 23 and has RNA ligase activity.

[0010] Furthermore, the non-natural RNA includes a neck-loop structure and a complementary region, and the substrate connection site is located in the neck-loop structure and / or the complementary region of the non-natural tRNA.

[0011] Furthermore, the 3' end of the substrate is a hydroxyl group.

[0012] Furthermore, the substrates include a first substrate and other substrates, the 5' end of the first substrate forms the 5' end of the non-natural tRNA, and the 5' ends of the other substrates are phosphates.

[0013] Furthermore, the number of the non-natural nucleotides in the substrate is ≥2.

[0014] Furthermore, the nucleotides at the 5' end and / or the 3' end of the substrate are non-natural nucleotides.

[0015] Furthermore, the substrate consists of non-natural nucleotides.

[0016] Furthermore, the number of substrates is 2-6; the length of the substrate is 15-60 nt.

[0017] Furthermore, the biosynthesis method includes: substrate annealing and substrate ligation; substrate annealing includes: dissolving the substrate in an annealing buffer to form an annealing system, and annealing the annealing system according to an annealing program, the annealing program including: heating the annealing system to 90°C, keeping warm for 1 minute, then lowering the temperature to 20°C at a cooling rate of 0.1°C per minute and then warming for 10 minutes to obtain an annealing product; substrate ligation includes: configuring the annealing product, RNA ligase and ATP into a ligation system to perform substrate ligation.

[0018] Furthermore, the annealing buffer comprises: 1mM to 10M NaCl, 1mM to 10M Tris-HCl, pH 4 to 10, 0.1mM to 10M EDTA; the reaction temperature of the substrate ligation is 0 to 70°C, and the reaction time is 10min to 100h; the concentration of the annealing product is 10μM to 2mM, the concentration of the RNA ligase is 0.02 to 4mg / mL, and the molar ratio of ATP to the annealing product is 1 to 10:1.

[0019] Furthermore, the connection system also includes MgCl2 and dithiothreitol; wherein the concentration of MgCl2 is 0.1mM~200M, and the concentration of dithiothreitol is 0.1mM~10M.

[0020] 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′-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.

[0021] To achieve the above object, according to a second aspect of the present invention, a non-natural tRNA is provided, wherein the non-natural tRNA includes the non-natural tRNA prepared by the above biosynthesis method.

[0022] Furthermore, the above-mentioned tRNA includes non-natural tRNA prepared using the above-mentioned biosynthesis method.

[0023] By applying the technical solution of the present invention, any one or more enzymes in the RNA ligase family Rnl1, Rnl2, Rnl3, and Rnl5 are used to biosynthesize tRNA. The biosynthesis method can overcome the problem of low tRNA yield in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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:

[0025] FIG1 shows a schematic structural diagram of a non-natural tRNA according to an embodiment of the present invention.

[0026] FIG2 shows the PAGE electrophoresis result of the ligation product of substrate 1 and substrate 2 according to Example 2 of the present invention.

[0027] FIG3 shows the UPLC result of the ligation product of substrate 3 and substrate 4 according to Example 3 of the present invention.

[0028] FIG4 shows the UPLC result of the ligation product of substrate 3 and substrate 4 according to Example 4 of the present invention.

[0029] FIG5 shows the PAGE electrophoresis result of the ligation product of substrate 9 and substrate 10 according to Example 6 of the present invention.

[0030] FIG6 shows the UPLC result of the ligation product of substrate 11 and substrate 12 according to Example 7 of the present invention. DETAILED DESCRIPTION

[0031] 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.

[0032] As mentioned in the background, there is currently very little research on tRNA synthesis. The main synthetic pathway is derived from natural tRNA synthesis within cells, which is then extracted to obtain tRNA, resulting in extremely low yields and susceptibility to nuclease contamination. In this application, the inventors attempted to explore a tRNA biosynthesis method, achieving efficient tRNA preparation and thus proposing a series of protection schemes in this application.

[0033] In a first typical embodiment of the present application, a method for the biosynthesis of tRNA is provided, which comprises: using RNA ligase to connect the 3′ end and 5′ end of different substrates to form a phosphodiester bond to form tRNA, where the number of substrates is ≥2; the RNA ligase comprises any one or more enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3 and Rnl5.

[0034] In a preferred embodiment, the substrate includes one or more non-natural nucleotides, and the tRNA includes a non-natural tRNA.

[0035] In the process of using tRNA to prepare nucleic acid drugs, because natural RNA is easily recognized and cleaved by intracellular nucleases, most nucleic acid drugs undergo artificial non-natural modifications to enhance their resistance to nucleases, thereby increasing specificity and reducing side effects. The prior art has not reported on the synthesis of non-natural tRNA. However, the biosynthetic method of the present application can synthesize not only natural tRNA but also non-natural tRNA.

[0036] 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: 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, SEQ ID NO: 22 or SEQ ID NO: 23; or a RNA ligase with SEQ ID NO: 1 to SEQ ID NO: 6 or SEQ ID NO: 10 to SEQ ID NO: 24. An enzyme having 70% or more identity with any of the RNA ligases shown in NO: 23, 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, and a protein having RNA ligase activity.

[0037] In the above-mentioned biosynthetic method, the above-mentioned RNA ligase can be used to connect substrates containing non-natural nucleotides and form tRNAs with higher-order structures. In the above-mentioned biosynthetic method, because the target product tRNA has a relatively complex higher-order structure and is a non-natural tRNA containing chemical modifications, the inventors screened and obtained the above-mentioned catalytically active RNA ligase from a variety of RNA ligases. The above-mentioned RNA ligase not only has the activity of catalyzing the ligation of RNA fragments containing non-natural nucleotides, but also has an active pocket that is structurally compatible with tRNA, capable of catalyzing the ligation of nicks between substrate RNA fragments in tRNA via phosphodiester bonds, thereby forming a non-natural tRNA with a higher-order structure.

[0038] 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.

[0039] 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).

[0040] 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).

[0041] 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:

[0042] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0043] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0044] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0045] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0046] 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.

[0047] In a preferred embodiment, the non-natural tRNA comprises a neck-loop structure and a complementary region, and the substrate binding site is located in the neck-loop structure and / or the complementary region of the non-natural tRNA.

[0048] In a preferred embodiment, the 3' end of the substrate is a hydroxyl group.

[0049] In a preferred embodiment, the substrates include a first substrate and other substrates, the 5' end of the first substrate forms the 5' end of the non-natural tRNA, and the 5' end of the other substrates is phosphate.

[0050] There are fragments capable of base complementary pairing between or inside the above-mentioned substrates. During the biosynthetic reaction, such fragments capable of base complementary pairing combine with each other to form the complementary region of the non-natural tRNA (including complementary region 1, complementary region 2, complementary region 3 and complementary region 4). After this base complementary pairing is completed, the high-level structure of the non-natural tRNA is initially formed, and the different substrates are connected by hydrogen bonds formed by base complementarity. The head and tail bases of different substrates are not connected to each other, and there are nicks. The above-mentioned nicks, that is, the connection sites of the substrates, are located in the neck loop structure (including neck loop 1, neck loop 2 and neck loop 3) and / or the complementary region of the non-natural tRNA. The active pocket of the above-mentioned RNA ligase can be adapted to the three-dimensional structure of the non-natural tRNA, and the catalytic activity is not affected by the three-dimensional structure. The connection catalytic substrates are connected by phosphodiester bonds, thereby repairing the nicks located on the non-natural tRNA. The structural schematic diagram of the above-mentioned non-natural tRNA is shown in Figure 1.

[0051] In a preferred embodiment, the number of non-natural nucleotides in the substrate is ≥2; preferably, the nucleotides at the 5′ end and / or 3′ end of the substrate are non-natural nucleotides; preferably, the substrate consists of non-natural nucleotides.

[0052] Non-natural nucleotides (xeno-nucleic acids, XNAs) are a class of nucleic acid molecules with non-natural backbones or nucleobases. Non-natural ribonucleotides are ribonucleotides with non-natural backbones or nucleobases. The aforementioned substrate donor contains non-natural nucleotides, and the tRNA prepared for ligation also contains non-natural nucleotides, thus representing a non-natural tRNA. If the 3′-terminal base of the substrate donor and / or the 5′-terminal base of the substrate acceptor are non-natural bases, the aforementioned RNA ligase can still perform ligation activity, even though the bases at the ligation site are different from natural bases and are artificially created non-natural bases.

[0053] In a preferred embodiment, the number of substrates is 2-6, including 2, 3, 4, 5 and 6. If the number of substrates is too large, the ligation efficiency and substrate conversion rate are low.

[0054] In a preferred embodiment, the length of the substrate is 15-60 nt, including but not limited to 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 nt.

[0055] In a preferred embodiment, the reaction conditions of the biosynthesis method include: substrate annealing and substrate ligation; substrate annealing includes: dissolving the substrate in an annealing buffer to form an annealing system, and annealing the annealing system according to an annealing program, the annealing program including: heating the annealing system to 90°C, keeping it warm for 1 minute, then reducing the temperature to 20°C at a rate of 0.1°C per minute, and then incubating for 10 minutes to obtain an annealed product; substrate ligation includes: configuring the annealing product, RNA ligase, and ATP into a ligation system, and performing substrate ligation.

[0056] Preferably, the annealing buffer comprises: 1 mM to 10 M NaCl, 1 mM to 10 M Tris-HCl, pH 4 to 10, and 0.1 mM to 10 M EDTA; more preferably, the annealing buffer comprises: 10 to 500 mM NaCl, 1 to 1000 mM Tris-HCl, pH 5 to 9, and 0.1 to 10 mM EDTA; preferably, the reaction temperature for substrate ligation is 0 to 70° C., and the reaction time is 10 min to 100 h; more preferably, the reaction temperature for substrate ligation is 0 to 50° C., and the reaction time is 0.2 to 72 h; preferably, the concentration of the annealing product is 10 μM to 2 mM, the concentration of the RNA ligase is 0.02 to 4 mg / mL, and the molar ratio of ATP to the annealing product is 1 to 10:1; Preferably, the reaction system further comprises MgCl2 and dithiothreitol; preferably, the concentration of MgCl2 is 0.1 mM to 200 M, and the concentration of dithiothreitol is 0.1 mM to 10 M; more preferably, the concentration of MgCl2 is 1 to 20 mM, and the concentration of dithiothreitol is 0.1 to 10 mM.

[0057] In the annealing buffer, the concentration of NaCl includes but is not limited to 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, 1 M, 2 M, 3 M, 5 M, 7 M or 10 M; the concentration of Tris-HCl includes but is not limited to 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, 1 M, 2 M, 3 M, 5 M, 7 M or 10 M; the pH includes but is not limited to 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10; the concentration of EDTA includes but is not limited to 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, 1 M, 2 M, 3 M, 5 M, 7 M or 10 M. The concentration of MgCl includes, but is not limited to, 0.1 M, 0.2 M, 0.3 M, 0.5 M, 1 M, 2 M, 3 M, 5 M, 10 M, 20 M, 30 M, 40 M, 50 M, 70 M, 100 M, 150 M, or 200 M; the concentration of dithiothreitol includes, but is not limited to, 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM, 1 M, 2 M, 3 M, 5 M, 7 M, or 10 M.

[0058] The reaction temperature includes but is not limited to 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C or 70°C, and the reaction time includes but is not limited to 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 7 h, 10 h, 15 h, 20 h, 30 h, 40 h, 50 h, 60 h, 70 h, 80 h, 90 h or 100 h.

[0059] In a preferred embodiment, the non-natural nucleotides include: ribonucleotides having 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′-fluoro 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.

[0060] Among them, the locked nucleic acid is modified to Hexitol nucleic acid modification 2′-methoxyethyl modified 2′-Fluoro 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.

[0061] In a second typical embodiment of the present application, a tRNA is provided, which includes the tRNA prepared using the above-mentioned biosynthesis method.

[0062] In a preferred embodiment, the tRNA includes a non-natural tRNA prepared using the above-mentioned biosynthetic method.

[0063] The sequences, sources and families of the ligases used in this application are as follows.

[0064] Ligase L-1, SEQ ID NO: 1, Escherichia phage JN02, RNA ligase 1 (RNA ligase family Rnl1):

[0065] Ligase L-2, SEQ ID NO: 2, Bacteriophage AR1, RNA ligase 1:

[0066] Ligase L-3, SEQ ID NO: 3, Bacteriophage dhaeg, RNA ligase 2 (RNA ligase family Rnl2):

[0067] Ligase L-4, SEQ ID NO: 4, Vibrio phage, RNA ligase 1:

[0068] Ligase L-5, SEQ ID NO: 5, Archaea, RNA ligase 3 (RNA ligase family Rnl3):

[0069] Ligase L-6, SEQ ID NO: 6, fungus Deinococcus radiodurans, RNA ligase 5 (RNA ligase family Rnl5):

[0070] Ligase L-7, SEQ ID NO: 7, Trypanosoma brucei brucei, RNA ligase 2:

[0071] Ligase L-8, SEQ ID NO: 8, Acidobacteria bacterium, RNA ligase 1:

[0072] Ligase L-9, SEQ ID NO: 9, fungus Magnaporthe grisea, RNA ligase 5:

[0073] Ligase L-10, SEQ ID NO: 10, Klebsiella phage KP15, RNA ligase 2:

[0074] Ligase L-11, SEQ ID NO: 11, Vibrio phage, RNA ligase 1:

[0075] Ligase L-12, SEQ ID NO: 12, Bacteriophage, RNA ligase 2:

[0076] Ligase L-13, SEQ ID NO: 13, bacteria, RNA ligase 2:

[0077] Ligase L-14, SEQ ID NO: 14, Vibrio phage phi-ST2, RNA ligase 2:

[0078] Ligase L-15, SEQ ID NO: 15, Vibrio phage JN02, RNA ligase 2:

[0079] Ligase L-16, SEQ ID NO: 16, Vibrio phage JS98, RNA ligase 2:

[0080] Ligase L-17, SEQ ID NO: 17, Trypanosoma brucei gambiense, RNA ligase 2:

[0081] Ligase L-18, SEQ ID NO: 18, Bacteriophage RB69, RNA ligase 1:

[0082] Ligase L-19, SEQ ID NO: 19, Bacteriophage, RNA ligase 1:

[0083] Ligase L-20, SEQ ID NO: 20, bacterial, RNA ligase 3:

[0084] Ligase L-21, SEQ ID NO: 21, Klebsiella phage KP179, RNA ligase 1:

[0085] Ligase L-22, SEQ ID NO: 22, Vibrio phage nt-1 rnlA, RNA ligase 2:

[0086] Ligase L-23, SEQ ID NO: 23, Vibrio phage phi-ST2, RNA ligase 2:

[0087] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0088] Example 1 Purification and preparation of RNA ligase

[0089] The ligase protein was codon-optimized for recombinant expression in Escherichia coli. The optimized codons were synthesized to obtain the target gene. The target gene was then constructed into the pET28a(+) expression vector using NcoI and XhoI restriction sites, with a 10-his tag at the N-terminus. The recombinant plasmid containing the ligase gene was transformed into the cloning strain DH5a and cultured overnight on LB medium supplemented with 50 μg / mL kanamycin and 1.5% agar to obtain a single clone. The sequence of the clone was confirmed by sequencing. The single clone was picked and transferred to liquid LB medium supplemented with 50 μg / mL kanamycin, cultured at 37°C for 16 hours, centrifuged, and the supernatant discarded to obtain a bacterial slurry. The resulting slurry was then used to extract the plasmid using a plasmid extraction kit. The resulting plasmid was then transformed into the BL21(DE3) expression strain and cultured overnight on LB medium supplemented with 50 μg / mL kanamycin and 1.5% agar to obtain a single clone. The obtained monoclonal clone was picked into 5mL of LB medium containing 50μg / mL of kanamycin and cultured at 37℃ for 16h to obtain seed liquid. The seed liquid was inoculated into 500mL of LB medium (containing 50μg / mL of kanamycin) at a 0.1% inoculum, cultured at 37℃ until OD=0.6, induced by adding 0.1M IPTG, the culture temperature was adjusted to 20℃, and induced at 220rpm for 16h to obtain the induced expression of the ligase Escherichia coli bacterial solution. The bacterial solution was centrifuged at 8000rpm, and the supernatant was discarded to obtain the induced expression of Escherichia coli bacterial mud. 10mM imidazole was added to the crude enzyme solution, and the enzyme solution obtained after filtering with a 0.22μm filter membrane was purified with a nickel column. After desalting and changing the liquid, it was purified again with a strong anion column. The obtained enzyme solution was desalted and changed the liquid and stored in 30% glycerol.

[0090] Example 2 RNA ligation catalyzed synthesis of natural tRNA

[0091] Substrates 1 and 2 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 200 μM and slowly annealed. The annealing program was to heat the system to 90°C for 1 minute, then decrease the temperature to 20°C at a rate of 0.1°C / min and incubate for 10 minutes. The sequences of substrates 1 and 2 are shown in Table 1.

[0092] Add the annealed substrates 1 and 2 to a clean reagent bottle and prepare a reaction system to a final volume of 100 μL. The concentrations of substrates 1 and 2 are 100 μM, ATP is 0.4 mM, Tris-HCl (pH 7.5 @ 25°C) is 50 mM, MgCl2 is 10 mM, DTT (dithiothreitol) is 1 mM, and ligase is 0.2 mg / mL. Incubate the reaction system at 16°C for 16 h. The resulting reaction system was inactivated by heating the ligase at 80°C for 5 minutes, and the precipitate was removed by centrifugation at 12,000 rpm. Mass spectrometry confirmed the formation of the product. The molecular weight detected in the reaction systems containing ligases L-1, L-2, L-3, L-4, L-5, L-6, L-11, L-18, L-19, L-20, L-21, L-22, and L-23 was 23,571 ± 2, compared to the theoretical molecular weight of 23,568 ± 5, indicating the formation of tRNA product. No product molecular weight was detected in the reaction systems containing L-7, L-8, and L-9, indicating the absence of tRNA product. PAGE gel detection was performed on some reaction systems, and the electrophoresis diagram is shown in Figure 2, wherein lane 1 is a reaction system catalyzed by ligase L-9, lane 2 is a reaction system catalyzed by ligase L-1, lane 3 is a reaction system catalyzed by ligase L-2, lane 4 is a negative control group without the addition of ligase, lane 5 is a reaction system catalyzed by ligase L-3, lane 6 is a reaction system catalyzed by ligase L-11, lane 7 is a reaction system catalyzed by ligase L-7, and lane 8 is a reaction system catalyzed by ligase L-8.

[0093] Due to the large fragment lengths, PAGE gel analysis failed to effectively distinguish the two substrates. Substrate 1 and Substrate 2 elute at the same position, resulting in a single nucleic acid band in the unenzymed reaction system, a mixed band of Substrate 1 and Substrate 2. Furthermore, no new peaks were observed in the reaction systems of L-7, 8, and 9, while new peaks were observed in the system where the product molecular weight was detected, demonstrating the feasibility of the experimental design. The nick in the aforementioned connection is located in the tRNA neck-loop structure.

[0094] Table 1

[0095] In the present application, P represents the phosphate group on the 5th carbon atom of the 5'-terminal nucleotide.

[0096] Example 3 RNA ligation-catalyzed synthesis of non-natural tRNA

[0097] Substrates 3 and 4 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 200 μM and slowly annealed. The annealing program was to heat the system to 90°C for 1 minute, then decrease the temperature to 20°C at a rate of 0.1°C / min and incubate for 10 minutes. The sequences of substrates 3 and 4 are shown in Table 2.

[0098] Annealed substrates 3 and 4 were added to a clean reagent vial to prepare a reaction system with a final volume of 100 μL. The concentrations of substrates 3 and 4 were 100 μM, ATP was 0.4 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 the ligase at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. The product was identified by mass spectrometry. The molecular weight of the reaction system for ligases L1, L2, L3, L4, L5, L6, L-11, L-18, L-19, L-20, L-21, L-22, and L-23 was 24,530 ± 2, compared to the theoretical molecular weight of 24,526 ± 5. This indicates that a non-natural tRNA product was generated. No product molecular weight was detected in the reaction systems of L-7, L-8, and L-9, indicating that no tRNA product was generated. UPLC detection of the reaction system of L-1 revealed the generation of a new peak, demonstrating the feasibility of the experimental scheme. The UPLC results are shown in Figure 3. Using UPLC to detect the reaction system, a new peak with an elution time of 25.822 min was found. Combined with the mass spectrometry results, the new peak was confirmed to be the product. The nick position of the above connection is in the neck loop structure of tRNA.

[0099] Table 2

[0100] The m before A, C, G or U represents a 2' methoxy modification of the ribonucleotide, and P represents a phosphate group on the 5th C atom of the 5' terminal nucleotide.

[0101] All ribonucleotides of substrates 3 and 4 have a 2' methoxy modification.

[0102] Example 4 RNA ligation-catalyzed synthesis of non-natural tRNA

[0103] Substrates 5 and 6 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 200 μM and slowly annealed. The annealing program was to heat the system to 90°C for 1 minute, then decrease the temperature to 20°C at a rate of 0.1°C / min and incubate for 10 minutes. The sequences of substrates 5 and 6 are shown in Table 3.

[0104] The annealed substrates 5 and 6 were added to a clean reagent bottle (, and the reaction system was prepared to make the final volume 100 μL, the concentrations of substrates 5 and 6 were 100 μM, the ATP concentration was 0.4 mM, the Tris-HCl (pH 7.5@25°C) concentration was 50 mM, the MgCl2 concentration was 10 mM, the DTT concentration was 1 mM, and the ligase concentration was 0.2 mg / mL. The reaction system was reacted at 16°C for 16 h. The obtained reaction system was heated at 80°C. The ligase was inactivated for 5 minutes, and the precipitate was removed by centrifugation at 12,000 rpm. Mass spectrometry was used to identify the product. The molecular weight detected in the reaction systems of ligases L1, L2, L3, L4, L5, L6, L-11, L-18, L-19, L-20, L-21, L-22, and L-23 was 24,171 ± 2, compared to the theoretical molecular weight of 24,166 ± 5. This indicates the formation of a non-natural tRNA product. No product molecular weight was detected in the reaction systems of L-7, L-8, and L-9, indicating the absence of tRNA product. UPLC analysis of the reaction system of L-1 revealed the formation of a new peak, demonstrating the feasibility of the experimental scheme. The UPLC results are shown in Figure 4. UPLC analysis of the reaction system revealed the formation of a new peak with an elution time of 20.512 minutes. Combined with the mass spectrometry results, this new peak was confirmed to be the product. The nick in the aforementioned ligation is located within the tRNA neck loop structure.

[0105] Table 3

[0106] Wherein, m before A, C, G or U represents a 2′-methoxy modification of the ribonucleotide, f represents a 2′-fluorine modification of the ribonucleotide, r represents a 2′-hydroxyl modification of the ribonucleotide (i.e., an unmodified ribonucleotide), and P represents a phosphate group on the 5th C atom of the 5′-terminal nucleotide.

[0107] The substrate 5 has 2' methoxy modifications on the 1st and 3rd ribonucleotides, 2' fluorine modifications on the 2nd, 36th and 37th ribonucleotides, and unmodified ribonucleotides on positions 4-35.

[0108] The ribonucleotides at positions 1, 2, 33, and 34 of substrate 6 have 2' fluorine modifications, and positions 3-32 and 35-37 are unmodified ribonucleotides.

[0109] Example 5 RNA ligation-catalyzed synthesis of non-natural tRNA

[0110] Substrates 7 and 8 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 200 μM and slowly annealed. The annealing program was to heat the system to 90°C for 1 minute, then decrease the temperature to 20°C at a rate of 0.1°C / min and incubate for 10 minutes. The sequences of substrates 7 and 8 are shown in Table 4.

[0111] Annealed substrates 7 and 8 were added to a clean reagent vial to prepare a reaction system with a final volume of 100 μL. The concentrations of substrates 7 and 8 were 100 μM, ATP was 0.4 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 the ligase at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. The product was identified by mass spectrometry. The molecular weight of the reaction system for ligases L1, L2, L3, L4, L5, L6, L-11, L-18, L-19, L-20, L-21, L-22, and L-23 was 26,367 ± 2, compared to the theoretical molecular weight of 26,364 ± 5. This indicates the formation of a non-natural tRNA product. No product molecular weight was detected in the reaction systems of L-7, L-8, and L-9, indicating that no tRNA product was generated. The nick in the aforementioned connection is located within the tRNA neck loop structure.

[0112] Table 4

[0113] Wherein, m before A, C, G or U represents a 2′-methoxy modification of the ribonucleotide, r represents a 2′-hydroxyl modification of the ribonucleotide (i.e., an unmodified ribonucleotide), s represents a phosphorothioate modification, and P represents a phosphate group on the 5th C atom of the 5′-terminal nucleotide.

[0114] Substrate 7 has 2' methoxy modifications on all ribonucleotides and phosphorothioate modifications on ribonucleotides 2-37.

[0115] The substrate 8 has 2'-methoxy modification on the 1st to 35th ribonucleotides, phosphorothioate modification on the 1st to 35th ribonucleotides, and unmodified ribonucleotides on the 36th to 38th ribonucleotides.

[0116] Example 6 RNA ligation catalyzed synthesis of natural tRNA

[0117] Substrates 9 and 10 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 200 μM and slowly annealed. The annealing program was to heat the system to 90°C for 1 minute, then decrease the temperature to 20°C at a rate of 0.1°C / min and incubate for 10 minutes. The sequences of substrates 9 and 10 are shown in Table 5.

[0118] Annealed substrates 9 and 10 were added to a clean reagent vial, and the reaction system was prepared to a final volume of 100 μL. The concentrations of substrates 9 and 10 were 100 μM, ATP was 0.4 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 the ligase at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. The product was identified by mass spectrometry. The molecular weight of the reaction system containing ligases L1, L2, L3, L4, L5, L6, L10, L11, L12, L13, L14, L15, L16, and L17 was 23,560.3, compared to the theoretical molecular weight of 23,564 ± 5, indicating the generation of native tRNA products. No product molecular weight was detected in the reaction systems of L-7, L-8, and L-9, indicating that no tRNA product was generated. PAGE gel detection was performed on some of the reaction samples above, and the electrophoresis results are shown in Figure 5. It was found that new bands were generated in the reaction system, while no bands were generated in the system without mass spectrometry product response, which was consistent with the negative control reaction bands without enzyme addition. The nick position of the above connection is in the complementary region of tRNA. In Figure 5, lane 1 is a negative control group without the addition of ligase, lane 2 is a reaction system catalyzed by ligase L-10, lane 3 is a reaction system catalyzed by ligase L-11, lane 4 is a reaction system catalyzed by ligase L-12, lane 5 is a reaction system catalyzed by ligase L-1, lane 6 is a reaction system catalyzed by ligase L-2, and lane 7 is a reaction system catalyzed by ligase L-7.

[0119] Table 5

[0120] P represents the phosphate group on the 5th carbon atom of the 5'-terminal nucleotide.

[0121] Example 7 RNA ligation-catalyzed synthesis of non-natural tRNA

[0122] Substrates 11 and 12 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 200 μM and slowly annealed. The annealing program was to heat the system to 90°C for 1 minute, then decrease the temperature to 20°C at a rate of 0.1°C / min and incubate for 10 minutes. The sequences of substrates 11 and 12 are shown in Table 6.

[0123] Annealed substrates 11 and 12 were added to a clean reagent vial to prepare a reaction system with a final volume of 100 μL. The concentrations of substrates 11 and 12 were 100 μM, ATP was 0.4 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 the ligase at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. The product was identified by mass spectrometry. The molecular weight of the reaction system for ligases L1, L2, L3, L4, L5, L6, L10, L11, L12, L13, L14, L15, L16, and L17 was 24,601.6, compared to the theoretical molecular weight of 24,606.2 ± 5. This indicates that a non-natural tRNA product was generated. No product molecular weight was detected in the reaction systems of L-7, L-8, and L-9, indicating that no tRNA product was generated. UPLC was used to detect product generation in the reaction system of L1, and the results showed the generation of a new peak, indicating that the technology is feasible. The UPLC results are shown in Figure 6. The reaction system was tested using UPLC, and a new peak with an elution time of 21.045 min was found. Combined with the mass spectrometry results, the new peak was confirmed to be the product. The nick position of the above connection is in the complementary region of tRNA.

[0124] Table 6

[0125] The m before A, C, G or U represents a 2' methoxy modification of the ribonucleotide, and P represents a phosphate group on the 5th C atom of the 5' terminal nucleotide.

[0126] All ribonucleotides in substrates 11 and 12 have 2' methoxy modifications.

[0127] Example 8 RNA ligation-catalyzed synthesis of non-natural tRNA

[0128] To attempt to synthesize tRNA using more fragments, substrates 13, 14, and 15 were dissolved in annealing buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a final concentration of 300 μM and slowly annealed. The annealing procedure consisted of heating the system to 90°C for 1 minute, then decreasing the temperature to 20°C at a rate of 0.1°C per minute and incubating for 10 minutes. The sequences of substrates 13, 14, and 15 are shown in Table 7.

[0129] Annealed substrates 13, 14, and 15 were added to a clean reagent vial, and the reaction system was prepared to a final volume of 100 μL. The concentrations of substrates 13, 14, and 15 were 100 μM, ATP was 0.4 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 the ligase at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. Mass spectrometry was used to identify the product. The molecular weight detected in the reaction systems of ligases L1, L2, and L4 was 24,601.6, compared to the theoretical molecular weight of 24,606.2 ± 5, indicating the generation of a non-natural tRNA product. The nick position of the ligation is located in the neck-loop structure of the tRNA.

[0130] Table 7

[0131] The m before A, C, G or U represents a 2' methoxy modification of the ribonucleotide, and P represents a phosphate group on the 5th C atom of the 5' terminal nucleotide.

[0132] All ribonucleotides of substrates 13, 14, and 15 have 2' methoxy modifications.

[0133] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the present invention has discovered that some RNA ligases can achieve the biosynthesis of non-natural tRNA containing non-natural nucleotides, and can be used for the biosynthesis of tRNA and even non-natural tRNA. Utilizing this biosynthesis method, tRNA can be prepared efficiently with high product yield and purity, and non-natural tRNA that is difficult to synthesize in the prior art can be prepared.

[0134] 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 shall be included within the scope of protection of the present invention.

Claims

1. A method for biosynthesis of tRNA, characterized in that: The biosynthetic method comprises: Using RNA ligase to connect the 3′ end and the 5′ end of different substrates to form a phosphodiester bond to form the tRNA, wherein the number of the substrates is ≥2; The RNA ligase includes any one or more enzymes of the RNA ligase family Rnl1, Rnl2, Rnl3 and Rnl5.

2. The biosynthesis method according to claim 1, characterized in that The substrate includes one or more non-natural nucleotides, and the tRNA includes non-natural tRNA.

3. The biosynthesis method according to claim 2, 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: 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, SEQ ID NO: 22, or SEQ ID NO: 23; or A protein having 70% or more identity with any one of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 6 or SEQ ID NO: 10 to SEQ ID NO: 23 and having RNA ligase activity.

4. The biosynthesis method according to claim 2, characterized in that The non-natural tRNA comprises a neck loop structure and a complementary region, and the connection site of the substrate is located in the neck loop structure and / or the complementary region.

5. The biosynthesis method according to claim 2, characterized in that: The 3' end of the substrate is a hydroxyl group.

6. The biosynthesis method according to claim 2, characterized in that: The substrates include a first substrate and other substrates, wherein the 5′ end of the first substrate forms the 5′ end of the non-natural tRNA, and the 5′ end of the other substrates is phosphate.

7. The biosynthesis method according to claim 2, characterized in that: The number of non-natural nucleotides in the substrate is ≥2.

8. The biosynthesis method according to claim 7, characterized in that The nucleotides at the 5' end and / or 3' end of the substrate are the non-natural nucleotides.

9. The biosynthesis method according to claim 8, characterized in that The substrate consists of the non-natural nucleotide.

10. The biosynthesis method according to claim 2, characterized in that: The number of the substrates is 2-6; The length of the substrate is 15-60 nt.

11. The biosynthesis method according to claim 2, characterized in that: The biosynthetic method comprises: substrate annealing and substrate ligation; The substrate annealing comprises: dissolving the substrate in an annealing buffer to form an annealing system, annealing the annealing system according to an annealing program, wherein the annealing program comprises: heating the annealing system to 90° C., keeping the temperature for 1 minute, and then Then, the temperature was lowered to 20°C at a rate of 0.1°C per minute and then heated for 10 minutes to obtain an annealed product; The substrate connection includes: configuring the annealing product, the RNA ligase and ATP into a connection system to perform the substrate connection.

12. The biosynthesis method according to claim 11, characterized in that: The annealing buffer comprises: 1 mM to 10 M NaCl, 1 mM to 10 M Tris-HCl, pH 4 to 10, and 0.1 mM to 10 M EDTA; The reaction temperature of the substrate connection is 0-70°C, and the reaction time is 10min-100h; The concentration of the annealing product is 10 μM to 2 mM, the concentration of the RNA ligase is 0.02 to 4 mg / mL, and the molar ratio of the ATP to the annealing product is 1 to 10:

1.

13. The biosynthesis method according to claim 12, characterized in that: The connection system also includes MgCl2 and dithiothreitol, wherein The concentration of MgCl2 is 0.1mM to 200M, and the concentration of dithiothreitol is 0.1mM to 10M.

14. The biosynthesis method according to claim 2, 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′-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.

15. A tRNA, characterized in that The tRNA includes the tRNA prepared by the biosynthetic method according to any one of claims 1 to 14.

16. The tRNA according to claim 15, characterized in that The tRNA includes the non-natural tRNA prepared by the biosynthetic method according to any one of claims 2 to 14.

Images