TRNA decoder for sequence-specific readthrough termination codon

Abstract

The invention provides a mutant tRNA (transfer ribonucleic acid) capable of specifically decoding a pseudouracil modified codon in a transcriptome and a translation group. In addition, the invention also provides a translation system containing the mutant tRNA, and a method for realizing site-specific amino acid insertion by responding to the pseudouracil modification codon based on the mutant tRNA. Furthermore, the invention also provides an RNA modification assisted codon expansion (RCE) technology based on the mutant tRNA.

Description

Technical Field

[0001] The present application relates to the field of synthetic biology, in particular to the fields of stop codon readthrough and protein engineering. In particular, the present application provides a mutant tRNA that sequence-specifically decodes pseudouracil-modified codons within the transcriptome and translation group. In addition, the present application also provides a translation system comprising the mutant tRNA, and a method for achieving site-specific amino acid insertion based on the mutant tRNA in response to the pseudouracil-modified codon. Further, the present application also provides an RNA modification-assisted codon expansion (RCE) technology based on the mutant tRNA. Background Art

[0002] Stop codons are universal translation termination signals in most cells, terminating the translation of a wide range of proteins across a wide range of organisms. Therefore, their function is highly conserved and rigorous, as failure to do so would result in a higher probability of translation errors in proteins, key executors of cellular life. In one scenario, premature termination codons (PTCs) can occur in physiologically important gene coding regions, preventing the normal processing and folding of essential proteins. In another, stop codons (SCRs) can read through the stop codons that signal gene translation termination, generating extended sequences at the protein's carboxyl terminus, thereby regulating protein localization and function. Therefore, in scenarios where PTCs occur, researchers have explored various approaches to read through these PTCs and restore expression of the proteins they contain. In the case of SCRs, researchers aim to ensure that the native stop codons function properly, minimizing the impact of exogenously introduced translational machinery.

[0003] In nature, there is pyrrolysyltRNA (pyrrolysine tRNA) from archaea that can read through the stop codon. Since the stop codon does not encode any amino acid, researchers have encoded the stop codon as a writable module based on pyrrolysyl tRNA from archaea. At the same time, through engineering modification to identify the corresponding PylRS (pyrrolysyl-tRNA synthetase), researchers can make pyrrolysine tRNA aminoacylated with different amino acids, and ultimately read the stop codon as the desired amino acid. This type of technology is called genetic code expansion (GCE). This technology not only breaks through the limitations of the natural amino acid skeleton, but also supports the modification of complex functional groups, providing more solutions for the precise analysis of the interactions of specific proteins. However, it should be noted that GCE technology requires a bioorthogonal system that simultaneously meets two conditions: first, the exogenously introduced orthogonal tRNA must maintain strict orthogonality with the aminoacyl-tRNA synthetase (aaRS) of the natural amino acid in the cell to avoid erroneous aminoacylation; second, the engineered aaRS must have absolute substrate specificity for the target ncAA, while completely excluding recognition interference from endogenous amino acids and host tRNA.

[0004] Based on this, Xia Qing's team used the PylRS / tRNAPyl orthogonal system to achieve readthrough of stop codons in a Duchenne muscular dystrophy mouse model (Shi, N., Yang, Q., Zhang, H. et al. Nat Biomed Eng 2022). However, GCE technology may cause abnormal extension of endogenous protein translation by recoding stop codons to the required amino acids. As a universal signal for translation termination, the highly conserved function of stop codons is crucial for maintaining the fidelity of protein synthesis. Therefore, it is urgent to develop a tRNA decoder with sequence specificity in both the transcriptome and the translation group to achieve sequence-specific readthrough of stop codons.

[0005] To balance readthrough efficiency and specificity, researchers have developed four types of orthogonalization strategies: (1) Quadruple codon system: designing orthogonal ribosome recognition quadruple codons to avoid competition with natural triplet codons, such as the successful simultaneous insertion of five p-azidophenylalanines (pAzF) into sfGFP by the Jewett and Mankin teams; (2) Codon reallocation: releasing specific codons through genome editing, such as the Church team's construction of a streamlined Escherichia coli system using only 57 codons, and the Lin Shixian laboratory's reprogramming of TCG codons to increase the production of ncAA integrins; (3) Non-natural base expansion: introducing artificial base pairs such as dNaM-dTPT3 to construct semi-synthetic organisms with a six-letter genetic code; (4) Phase separation membraneless organelles: using phase separation elements to isolate the target mRNA from the translation machinery to achieve spatially specific ncAA insertion.

[0006] While these strategies can partially reduce the risk of misinsertion of ncAAs, they all face the challenges of requiring multiple engineering modifications in mammalian cells, are technically complex, and may interfere with endogenous pathways. Therefore, based on current technological advancements, further development of simpler and more efficient strategies for genetic code expansion is needed. Summary of the Invention

[0007] RNA modifications can modulate the physicochemical properties of RNA molecules, resulting in different codon properties. Among them, pseudouracil (Ψ) has garnered significant attention due to its ability to enhance base pairing. However, a tRNA decoder specifically recognizing Ψ is currently lacking.

[0008] Based on in-depth research, the inventors of this application have developed a series of mutant tRNAs that can distinguish between Ψ-modified stop codons (ΨCodon, including ΨGA, ΨAG, and ΨAA) and unmodified stop codons during translation, thereby achieving specific decoding of ΨCodon. Furthermore, based on the mutant tRNA, the application also provides a translation system comprising the mutant tRNA, a method based on the mutant tRNA responding to the pseudouracil modified codon to achieve site-specific amino acid insertion, and an RNA modification-assisted codon expansion (RCE) technology based on the mutant tRNA.

[0009] 1. tRNA mutants

[0010] Therefore, in one aspect, the present application provides a tRNA mutant comprising a nucleotide substitution mutation at one or more positions selected from the group consisting of:

[0011] Position 27, position 28, position 29a, position 29b, position 30, position 31, position 32, position 33, position 37, position 38, position 39, position 40, position 41a, position 41b, position 41c, position 42, position 43.

[0012] Unless otherwise specified or clearly contradictory in context, when describing mutations contained in tRNAs in this application, the positions of the mutations are based on the internationally accepted standard tRNA numbering system (often referred to as "standard tRNA numbering" or "classical tRNA numbering"), as described in Ambrogelly A, et al. Pyrrolysine is not hardwired for cotranslational insertion at UAG codons. Proc Natl Acad Sci US A. 2007 Feb 27; 104(9): 3141-6. doi: 10.1073 / pnas.0611634104., the entire text of which is incorporated herein by reference.

[0013] The core of this numbering system is based on the crystal structure of yeast phenylalanine tRNA (solved in 1974) and is extended to all tRNA variants. According to the core rules of this standard tRNA numbering system:

[0014] tRNAs are numbered consecutively from 5' to 3', from positions 1 to 76. The functional regions are numbered as follows: acceptor stem (positions 1-7 and 66-72), D arm (positions 10-25), anticodon arm (positions 26-44, with the anticodon at positions 34-36), variable loop (positions 44-48, variable length), TΨC arm (positions 49-65), and 3'-CCA end (positions 73-76). When a tRNA inserts an extra nucleotide between standard positions X and X+1, the first inserted nucleotide is labeled Xa (e.g., between 41 and 42 → 41a), and subsequent insertions are labeled Xb, Xc, and so on.

[0015] Those skilled in the art are aware that, to accommodate the needs of codon expansion and diversification of unnatural amino acids, a variety of artificially engineered and / or screened tRNAs exist in the art, and the structures (e.g., sequence information) and functions of such tRNAs are known to those skilled in the art.

[0016] Based on the disclosure of this application, it will be readily understood by those skilled in the art that this application provides a series of tRNA mutations that are applicable to existing tRNAs (including not only natural tRNAs directly derived from organisms but also artificially modified / screened non-natural tRNAs) to impart a preference for recognizing Ψ-modified codons (e.g., ΨGA, ΨAG, ΨAA). Therefore, as used herein, the term "wild-type tRNA" includes not only natural tRNAs directly derived from organisms but also artificially modified / screened non-natural tRNAs (i.e., modified / screened non-natural tRNAs indirectly derived from organisms).

[0017] In certain embodiments, the tRNA mutant comprises one or more mutations selected from the group consisting of:

[0018] (1) 27G; (2) 28G or 28U; (3) 29aA or 29aU; (4) 29bA or 29bU; (5) 30U; (6) 31C, 31A, 31G or 31U; (7) 32U or 32A; (8) 33A or 33C; (9) 37A, 37G, 37U or 37C; (10) 38U or 38C; (11) 39A or 39C; (12) 40U; (13) 41aU or 41aG; (14) 41bC; (15) 41cA or 41cG; (16) 42A or 42C; (17) 43A, 42C or 43U.

[0019] As used herein, the mutation "27G" means that the nucleotide residue at position 27 (defined according to the standard tRNA numbering system described above) in the tRNA mutant is substituted with G; the mutation "28G" means that the nucleotide residue at position 28 (defined according to the standard tRNA numbering system described above) in the tRNA mutant is substituted with G.

[0020] Unless otherwise specified or clearly contradicted by the context, the meanings of other similar expressions herein shall be defined in a similar manner to the above.

[0021] In certain embodiments, the tRNA mutant comprises one or more mutations selected from the group consisting of:

[0022] (1) 31C, 31A, 31G or 31U; (2) 33A or 33C; (3) 37A, 37G, 37U or 37C; (4) 39A or 39C.

[0023] In certain embodiments, the tRNA mutant comprises a mutation selected from the group consisting of:

[0024] (1) 27G; (2) 28G or 28U; (3) 29aA or 29aU; (4) 29bA or 29bU; (5) 30U; (6) 31C, 31A, 31G, or 31U; (7) 32U or 32A; (8) 33A or 33C; (9) 37A, 37G, 37U, or 37C; (10) 38U or 38C; (11) 39A or 39C; (12) 40U; (13) 41aU or 41aG; (14) 41bC; (15) 41cA or 41cG; (16) 42A or 42C; (17) 43A, 42C or 43U; (18) 31C and 37C; (19) 31G and 37C; (20) 31U and 37C; (21) 30U and 37G; (22) 31C and 33C; (23) 31C and 37G; (24) 31G and 33C; (25) 31G and 37G; (26) 31U and 33C; (27) 31U and 37G; (28) 33C and 37G.

[0025] In certain embodiments, the tRNA mutant comprises a mutation selected from the group consisting of:

[0026] (1) 29aA or 29aU; (2) 29bA or 29bU; (3) 31C, 31A, 31G or 31U; (4) 33A or 33C; (5) 37A, 37G, 37U or 37C; (6) 39A or 39C; (7) 31C and 37C; (8) 31G and 37C; (9) 31U and 37C; (10) 31C and 33C; (11) 31C and 37G; (12) 31G and 33C; (13) 31G and 37G; (14) 31U and 33C; (15) 31U and 37G; (16) 33C and 37G.

[0027] In certain embodiments, the wild-type tRNA is selected from: tRNA derived from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), tRNAs derived from Methanosarcina spelaei Spe (Methanosarcina spelaei Spe) (e.g., SpetRNA Pyl ), tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), tRNAs derived from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ).

[0028] 1.1tRNA mutants-MmtRNA mutants

[0029] In certain embodiments, compared to a wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of:

[0030] (1) 30U; (2) 31C, 31G or 31U; (3) 33A or 33C; (4) 37G or 37C; (5) 38U or 38C; (6) 39A or 39C; (7) 40U;

[0031] In certain embodiments, compared to a wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of:

[0032] (1) 30U; (2) 31C, 31G or 31U; (3) 33A or 33C; (4) 37G or 37C; (5) 38U or 38C; (6) 39A or 39C; (7) 40U; (8) 31C and 37C; (9) 31G and 37C; (10) 31U and 37C; (11) 30U and 37G; (12) 31C and 33C; (13) 31C and 37G; (14) 31G and 33C; (15) 31G and 37G; (16) 31U and 33C; (17) 31U and 37G; (18) 33C and 37G.

[0033] In certain embodiments, compared to a wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of:

[0034] (1) 31C, 31G or 31U; (2) 33A or 33C; (3) 37G or 37C; (4) 39A or 39C; (5) 31C and 37C; (6) 31G and 37C; (7) 31U and 37C; (8) 31C and 33C; (9) 31C and 37G; (10) 31G and 33C; (11) 31G and 37G; (12) 31U and 33C; (13) 31U and 37G; (14) 33C and 37G.

[0035] In certain embodiments, the tRNA mutant comprises an anticodon stem-loop (ASL) sequence selected from any one of SEQ ID NOs: 148-169.

[0036] In certain embodiments, the tRNA mutant comprises a sequence as set forth below:

[0037] S1-S ASL -S2

[0038] wherein,

[0039] S1 comprises a sequence as set forth in SEQ ID NO: 170 or 172, or a variant thereof; S ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 148-169; S2 comprises a sequence as set forth in SEQ ID NO: 171 or 173, or a variant thereof;

[0040] wherein the variant has one or several nucleotide substitutions, deletions or additions (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) in comparison with the sequence from which it is derived.

[0041] In certain embodiments, the tRNA mutant comprises a sequence as set forth below:

[0042] S1-S ASL -S2

[0043] wherein,

[0044] (1) S1 comprises a sequence as set forth in SEQ ID NO: 170, or a variant thereof; S ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 148-169; S2 comprises a sequence as set forth in SEQ ID NO: 171, or a variant thereof; or, (2) S1 comprises a sequence as set forth in SEQ ID NO: 172, or a variant thereof; S ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 148-169; S2 comprises a sequence as set forth in SEQ ID NO: 173, or a variant thereof;

[0045] wherein the variant has one or several nucleotide substitutions, deletions or additions (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) in comparison with the sequence from which it is derived.

[0046] In certain embodiments, the wild-type tRNA derived from Methanosarcina mazei is MmtRNA Pyl .

[0047] In certain embodiments, MmtRNA Pyl comprising a sequence as shown in any one of SEQ ID NOs: 68, 144, and 147. In certain embodiments, the tRNA mutant comprises a sequence as shown below:

[0048] S1-S ASL -S2

[0049] in,

[0050] S1 comprises the sequence shown in SEQ ID NO: 170 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 148, 150-169; S2 comprises a sequence as shown in SEQ ID NO: 171 or a variant thereof; or S1 comprises a sequence as shown in SEQ ID NO: 172 or a variant thereof;

[0051] The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

[0052] In certain embodiments, the wild-type tRNA derived from Methanosarcina mazei is MmtRNA Tet .

[0053] In certain embodiments, MmtRNA Tet Comprising the sequence shown in SEQ ID NO:72.

[0054] In certain embodiments, compared to MmtRNA Tet , the tRNA mutant comprises mutation: 37G. In certain embodiments, the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 149. In certain embodiments, the tRNA mutant comprises the sequence shown below:

[0055] S1-S ASL -S2

[0056] in,

[0057] S1 comprises the sequence shown in SEQ ID NO: 172 or a variant thereof; ASLis an ASL sequence comprising a sequence selected from the group consisting of SEQ ID NO: 149; S2 comprises a sequence as shown in SEQ ID NO: 173 or a variant thereof;

[0058] The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

[0059] It is easy for those skilled in the art to understand that, in order to ensure the correct folding of tRNA and the stability of its structure, mature tRNA with biological activity usually has a high proportion of modified bases (e.g., T, Ψ, DHU and I and various methylated bases), and a "CCA" structure is connected to the 3' end. Such modifications are well known to those of ordinary skill in the art (for example, see Peschek J, Tuorto F. Interplay Between tRNA Modifications and Processing. J Mol Biol. 2025 Aug 15; 437(16): 169198. doi: 10.1016 / j.jmb.2025.169198. Epub 2025 May 22. PMID: 40404521.; the entire text of which is incorporated herein by reference). Therefore, based on the disclosure of this application, it is easy for those skilled in the art to understand that when describing the sequence of a tRNA or its ASL or the remaining segments, this application is intended to include not only the naked nucleotide sequence information corresponding to the sequence shown, but also the various tRNA modifications (e.g., modified bases, "CCA" tails) that may be included therein. For example, when describing a tRNA mutant "comprising a sequence selected from any one of SEQ ID NOs: 44-51, 53, 55-63," it includes not only tRNAs having the naked sequence information corresponding to the sequence shown in any one of SEQ ID NOs: 44-51, 53, 55-63, but also tRNAs having the naked sequence information corresponding to the sequence shown in any one of SEQ ID NOs: 44-51, 53, 55-63 and various modifications naturally occurring in tRNA (e.g., modified bases, "CCA" tails).

[0060] 1.1.1 tRNA mutant-MmtRNA mutant-ΨAG

[0061] In certain embodiments, the tRNA mutant recognizes ΨAG.

[0062] In certain embodiments, the tRNA mutant comprises the anticodon CUA.

[0063] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0064] (1) Compared with wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 30U; (ii) 31C, 31G, or 31U; (iii) 33A or 33C; (iv) 37G or 37C; (v) 38U or 38C; (v) 40U; In certain embodiments, compared to the wild-type tRNA derived from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of: (i) 30U; (ii) 31C, 31G, or 31U; (iii) 33A or 33C; (iv) 37G or 37C; (v) 38U or 38C; (vi) 40U; (vii) 31C and 37C; (viii) 31G and 37C; (ix) 31U and 37C; (x) 30U and 37G; (xi) 31C and 33C; (xii) 31C and 37G; (xiii) 31G and 33C; (xiv) 31G and 37G; (xv) 31U and 33C; (xvi) 31U and 37G; (xvii) 33C and 37G; in certain embodiments, compared to a wild-type tRNA derived from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of: (i) 31C, 31G, or 31U; (ii) 33A or 33C; (iii) 37G or 37C; (iv) 31C and 37C; (v) 31G and 37C; (vi) 31U and 37C; (vii) 31C and 33C; (viii) 31C and 37G; (ix) 31G and 33C; (x) 31G and 37G; (xi) 31U and 33C; (xii) 31U and 37G; (xiii) 33C and 37G;

[0065] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 116-123, 125, and 127-136;

[0066] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 44-51, 53, 55-63 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) compared to the sequence from which it is derived.

[0067] In certain embodiments, the wild-type tRNA derived from Methanosarcina mazei is MmtRNA Pyl .

[0068] In certain embodiments, the wild-type tRNA derived from Methanosarcina mazei is MmtRNA Tet .

[0069] 1.1.2 tRNA mutant-MmtRNA mutant-ΨGA

[0070] In certain embodiments, the tRNA mutant recognizes ΨGA.

[0071] In certain embodiments, the tRNA mutant comprises the anticodon UCA.

[0072] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0073] (1) Compared with wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 31C; (ii) 37G; (iii) 39A or 39C;

[0074] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 79-82;

[0075] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 7-10 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) compared to the sequence from which it is derived.

[0076] In certain embodiments, the wild-type tRNA derived from Methanosarcina mazei is MmtRNA Pyl .

[0077] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0078] (1) Compared with the mtRNA Pyl , the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 31C; (ii) 37G; (iii) 39A or 39C;

[0079] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 79-82 and 138;

[0080] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 7-10, 65, or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions, or additions) compared to the sequence from which it is derived.

[0081] In certain embodiments, the wild-type tRNA derived from Methanosarcina mazei is MmtRNA Tet .

[0082] In certain embodiments, compared to that derived from MmtRNA Tet , the tRNA mutant comprises the mutation: 37G. In certain embodiments, the tRNA mutant comprises an ASL sequence selected from the group consisting of SEQ ID NO: 138. In certain embodiments, the tRNA mutant comprises a sequence selected from the group consisting of SEQ ID NO: 65 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions, or additions) compared to the sequence from which it is derived.

[0083] 1.1.3 tRNA mutant-MmtRNA mutant-ΨAA

[0084] In certain embodiments, the tRNA mutant recognizes ΨAA.

[0085] In certain embodiments, the tRNA mutant comprises the anticodon UUA.

[0086] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0087] (1) Compared with wild-type tRNA from Methanosarcina mazei (e.g., MmtRNAPyl 、MmtRNA Tet ), the tRNA mutant comprises the mutation: 37G;

[0088] (2) the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 87;

[0089] (3) The tRNA mutant comprises a sequence selected from SEQ ID NO: 15 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) compared to the sequence from which it is derived.

[0090] 1.2 tRNA mutants-AlvtRNA mutants

[0091] In certain embodiments, the tRNA mutant is more highly expressed than a wild-type tRNA derived from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), comprising one or more mutations selected from the group consisting of:

[0092] (1) 28G or 28U; (2) 29aA or 29aU; (3) 29bA or 29bU; (4) 31C or 31A; (5) 32U or 32A; (6) 33A or 33C; (7) 37C; (8) 38U; (9) 41aU or 41aG.

[0093] In certain embodiments, compared to a wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), the tRNA mutant comprises a mutation selected from the group consisting of:

[0094] (1) 28G or 28U; (2) 29aA or 29aU; (3) 29bA or 29bU; (4) 37C.

[0095] In certain embodiments, the wild-type tRNA derived from Methanomethylophilus alvus is AlvtRNA Pyl .

[0096] In certain embodiments, AlvtRNA Pyl It comprises a sequence as shown in any one of SEQ ID NOs: 69, 142, and 145.

[0097] In certain embodiments, the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 174-188.

[0098] In certain embodiments, the tRNA mutant comprises the sequence shown below:

[0099] S1-S ASL -S2

[0100] in,

[0101] S1 comprises the sequence shown in SEQ ID NO: 189 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 174-188; S2 comprises a sequence as shown in SEQ ID NO: 190 or a variant thereof;

[0102] The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

[0103] 1.2.1 tRNA mutant-Alv tRNA mutant-ΨAG

[0104] In certain embodiments, the tRNA mutant recognizes ΨAG.

[0105] In certain embodiments, the tRNA mutant comprises the anticodon CUA.

[0106] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0107] (1) Compared with the wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 28G or 28U; (ii) 29aU; (iii) 29bA or 29bU; (iv) 31C or 31A; (v) 32A; (vi) 37C; (vii) 38U; (viii) 41aG; in certain embodiments, compared to a wild-type tRNA derived from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), wherein the tRNA mutant comprises a mutation selected from the group consisting of: (i) 28G or 28U; (ii) 29aU; (iii) 29bA or 29bU; (iv) 37C;

[0108] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 88-98;

[0109] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 16-26 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions, or additions) compared to the sequence from which it is derived.

[0110] 1.2.2 tRNA mutant-Alv tRNA mutant-ΨGA

[0111] In certain embodiments, the tRNA mutant recognizes ΨGA.

[0112] In certain embodiments, the tRNA mutant comprises the anticodon UCA.

[0113] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0114] (1) Compared with the wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 32U; (ii) 33A or 33C; (iii) 41aU;

[0115] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 73-76;

[0116] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 1-4 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions, or additions) compared to the sequence from which it is derived.

[0117] 1.2.3 tRNA mutant-Alv tRNA mutant-ΨAA

[0118] In certain embodiments, the tRNA mutant recognizes ΨAA.

[0119] In certain embodiments, the tRNA mutant comprises the anticodon UUA.

[0120] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0121] (1) Compared with the wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl), the tRNA mutant comprises the mutation: 29aA;

[0122] (2) the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 83;

[0123] (3) The tRNA mutant comprises a sequence selected from SEQ ID NO: 11 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) compared to the sequence from which it is derived.

[0124] 1.3tRNA mutants-InttRNA mutants

[0125] In certain embodiments, the tRNA mutant is more tRNA-specific than a wild-type tRNA derived from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), comprising one or more mutations selected from the group consisting of:

[0126] (1) 27G; (2) 28U; (3) 29aU; (4) 31U; (5) 33C; (6) 37A, 37G, or 37U; (7) 41aU; (8) 41bC; (9) 41cA or 41cG; (10) 42A or 42C; (11) 43A, 42C, or 43U.

[0127] In certain embodiments, compared to a wild-type tRNA derived from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises a mutation selected from the group consisting of:

[0128] (1) 28U; (2) 37A, 37G, or 37U; (3) 41aU; (4) 41bC; (5) 41cA or 41cG.

[0129] In certain embodiments, the wild-type tRNA derived from Methanomassiliicoccus intestinalis is InttRNA Pyl .

[0130] In certain embodiments, InttRNA Pyl It comprises the sequence shown in any one of SEQ ID NOs: 70, 143, and 146.

[0131] In certain embodiments, the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 191-209.

[0132] In certain embodiments, the tRNA mutant comprises the sequence shown below:

[0133] S1-S ASL -S2

[0134] in,

[0135] S1 comprises the sequence shown in SEQ ID NO: 210 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 191-209; S2 comprises a sequence as shown in SEQ ID NO: 211 or a variant thereof;

[0136] The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

[0137] 1.3.1 tRNA mutant-InttRNA mutant-ΨAG

[0138] In certain embodiments, the tRNA mutant recognizes ΨAG.

[0139] In certain embodiments, the tRNA mutant comprises the anticodon CUA.

[0140] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0141] (1) Compared with wild-type tRNA from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 27G; (ii) 28U; (iii) 29aU; (iv) 31U; (v) 33C; (vi) 37A, 37G, or 37U; (vii) 41aU; (viii) 41bC; (ix) 41cA or 41cG; (x) 42A or 42C; (xi) 43A, 43C, or 43U; in certain embodiments, compared to a wild-type tRNA derived from Methanomas siliicoccus intestinalis (e.g., InttRNA Pyl), wherein the tRNA mutant comprises a mutation selected from the group consisting of: (1) 28U; (2) 37G or 37U; (3) 41aU; (4) 41bC; (5) 41cA or 41cG;

[0142] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 99-115;

[0143] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 27-43 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions, or additions) compared to the sequence from which it is derived.

[0144] 1.3.2 tRNA mutant-InttRNA mutant-ΨGA

[0145] In certain embodiments, the tRNA mutant recognizes ΨGA.

[0146] In certain embodiments, the tRNA mutant comprises the anticodon UCA.

[0147] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0148] (1) Compared with wild-type tRNA from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises mutation: 37A or 37G;

[0149] (2) the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 77 or 78;

[0150] (3) The tRNA mutant comprises a sequence selected from SEQ ID NO: 5 or 6 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) compared to the sequence from which it is derived.

[0151] 1.3.3 tRNA mutant-InttRNA mutant-ΨAA

[0152] In certain embodiments, the tRNA mutant recognizes ΨAA.

[0153] In certain embodiments, the tRNA mutant comprises the anticodon UUA.

[0154] In certain embodiments, the tRNA mutant has one or more characteristics selected from the group consisting of:

[0155] (1) Compared with wild-type tRNA from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises the mutation: 37A, 37G or 37U;

[0156] (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 84-86;

[0157] (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 12-14 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions, or additions) compared to the sequence from which it is derived.

[0158] 1.4tRNA mutants-SpetRNA mutants

[0159] In certain embodiments, the tRNA mutant is more tRNA-dependent than a wild-type tRNA derived from Methanosarcina spelaei Spe (e.g., SpetRNA Pyl ), containing mutation: 37G.

[0160] In certain embodiments, the wild-type tRNA derived from Methanosarcina spelaei Spe is SpetRNA Pyl In certain embodiments, SpetRNA Pyl Comprising the sequence shown in SEQ ID NO: 66 or 71.

[0161] In certain embodiments, the tRNA mutant comprises an ASL sequence selected from the group consisting of SEQ ID NO: 212.

[0162] In certain embodiments, the tRNA mutant comprises the sequence shown below:

[0163] S1-S ASL -S2

[0164] in,

[0165] S1 comprises the sequence shown in SEQ ID NO: 213 or a variant thereof; ASLis an ASL sequence comprising a sequence selected from the group consisting of SEQ ID NO: 212; S2 comprises a sequence as shown in SEQ ID NO: 214 or a variant thereof;

[0166] The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

[0167] 1.4.1 tRNA mutants-SpetRNA mutants-ΨGA

[0168] In certain embodiments, the tRNA mutant recognizes ΨGA.

[0169] In certain embodiments, the tRNA mutant comprises the anticodon UCA.

[0170] In certain embodiments, the tRNA mutant comprises an ASL sequence selected from the group consisting of SEQ ID NO: 137.

[0171] In certain embodiments, the tRNA mutant comprises a sequence selected from SEQ ID NO: 64 or a variant thereof; wherein the variant has one or more nucleotide substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions) compared to the sequence from which it is derived.

[0172] In another aspect, the present application provides a nucleic acid molecule encoding a tRNA mutant as described above.

[0173] It is intended to be understood that the nucleic acid molecule may be a DNA molecule or an RNA molecule.

[0174] In another aspect, the present application provides a vector comprising the nucleic acid molecule described above.

[0175] In another aspect, the present application provides a host cell comprising the nucleic acid molecule or vector as described above.

[0176] In another aspect, the present application provides a composition comprising:

[0177] (a) a first component selected from: (i) a first orthogonal tRNA (O-tRNA), wherein the first O-tRNA is selected from the tRNA mutants described above; (ii) a nucleotide sequence encoding the first O-tRNA; (iii) any combination of (i) and (ii);

[0178] as well as,

[0179] (b) a second component selected from: (i) a first orthogonal aminoacyl-tRNA synthetase (O-RS) that recognizes a first amino acid and aminoacylates the first O-tRNA with the first amino acid; (ii) a nucleotide sequence encoding the first O-RS; or (iii) any combination of (i) and (ii).

[0180] In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the section "1.1 tRNA mutants-MmtRNA mutants". In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the section "1.2 tRNA mutants-AlvtRNA mutants".

[0181] In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the "1.3 tRNA mutants-InttRNA mutants" section.

[0182] In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the "1.4 tRNA mutants-SpetRNA mutants" section.

[0183] In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the sections “1.1.1 tRNA mutant-MmtRNA mutant-ΨAG”, “1.2.1 tRNA mutant-AlvtRNA mutant-ΨAG”, and “1.3.1 tRNA mutant-InttRNA mutant-ΨAG”.

[0184] In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the sections “1.1.2tRNA mutant-MmtRNA mutant-ΨGA”, “1.2.2tRNA mutant-AlvtRNA mutant-ΨGA”, “1.3.2tRNA mutant-InttRNA mutant-ΨGA”, and “1.4.1tRNA mutant-InttRNA mutant-ΨGA”.

[0185] In certain embodiments, the first O-tRNA is selected from the tRNA mutants described above in the sections “1.1.3 tRNA mutant-MmtRNA mutant-ΨAA”, “1.2.3 tRNA mutant-AlvtRNA mutant-ΨAA”, and “1.3.3 tRNA mutant-InttRNA mutant-ΨAA”.

[0186] In certain embodiments, the first amino acid is selected from natural amino acids and unnatural amino acids (e.g., Nε-benzyloxycarbonyl-L-lysine (CbzK), Nε-(cis-cyclooct-2- ene-l-oxycarbonyl)-L-lysine (TCOK), 3-(6-butyl-l,2,4,5-tetrazin-3-yl)-L-phenylalanine (TetBu), Nε-tert-butoxycarbonyl-L-lysine (BocK)).

[0187] In certain embodiments, the first amino acid is CbzK. In certain embodiments, the first O-RS is mmPylRS-306A384F (CbzK). In certain embodiments, the first O-RS comprises a sequence as set forth in SEQ ID NO: 139. In certain embodiments, the first O-tRNA is selected from the tRNA mutants of the “1.1 tRNA mutants - MmtRNA mutants” section above.

[0188] In certain embodiments, the first amino acid is TCOK. In certain embodiments, the first O-RS is mmPylRS-306A384F. In certain embodiments, the first O-RS comprises a sequence as set forth in SEQ ID NO: 139. In certain embodiments, the first O-tRNA is selected from the tRNA mutants of the “1.1 tRNA mutants - MmtRNA mutants” section above;

[0189] In certain embodiments, the first amino acid is CbzK. In certain embodiments, the first O-RS is 1R26PylRS (CbzK). In certain embodiments, the first O-RS comprises a sequence as set forth in SEQ ID NO: 140. In certain embodiments, the first O-tRNA is selected from the tRNA mutants of the “1.2 tRNA mutants - AlvtRNA mutants” section above.

[0190] In certain embodiments, the first amino acid is BocK. In certain embodiments, the first O-RS is RumEnPylRS (BocK). In certain embodiments, the first O-RS comprises a sequence as set forth in SEQ ID NO: 141. In certain embodiments, the first O-tRNA is selected from the tRNA mutants of the “1.3 tRNA mutants - InttRNA mutants” section above.

[0191] In certain embodiments, the first amino acid is TetBu. In certain embodiments, the first O-RS is TetBuRS. In certain embodiments, the first O-RS comprises a sequence as set forth in SEQ ID NO: 67. In certain embodiments, the first O-tRNA is MmtRNATet (UCA)-37G. In certain embodiments, the first O-tRNA comprises the sequence shown in SEQ ID NO:65.

[0192] In certain embodiments, the composition further comprises:

[0193] (I) a second O-tRNA, and / or a nucleotide sequence encoding the second O-tRNA; the second O-tRNA is selected from the tRNA mutants described above;

[0194] as well as,

[0195] (II) a second O-RS, and / or a nucleotide sequence encoding the second O-RS; wherein the second O-RS is capable of recognizing a second amino acid and aminoacylating the second O-tRNA with the second amino acid.

[0196] In certain embodiments, the second O-RS is capable of recognizing a second amino acid different from the first amino acid and aminoacylates the second O-tRNA with the second amino acid. In certain embodiments, the second O-tRNA recognizes a selector codon different from the selector codon recognized by the first O-tRNA.

[0197] In certain embodiments, the composition further comprises:

[0198] (iii) a third component selected from (i) an engineered guide RNA; (ii) a nucleotide sequence encoding the engineered guide RNA; (iii) any combination of (i) and (ii);

[0199] The engineered guide RNA comprises a backbone sequence and a guide sequence, wherein the guide sequence is capable of hybridizing with a target RNA, and the guide RNA is capable of guiding or recruiting a pseudouridine synthase (e.g., DKC1 protein), thereby modifying a target uridine residue in the target RNA hybridized with the engineered guide RNA into a pseudouridine residue.

[0200] In certain embodiments, the DKC1 protein comprises the sequence shown in SEQ ID NO:124.

[0201] In certain embodiments, the engineered guide RNA is an engineered guide small nucleolar RNA (gsnoRNA).

[0202] In certain embodiments, the backbone sequence of the engineered gsnoRNA is derived from wild-type H / ACA-snoRNAs: ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17.

[0203] In certain embodiments, the sequence of the engineered gsnoRNA is selected from: (a) a sequence as shown in SEQ ID NO: 215; (b) a sequence having one or more nucleotide residue substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residue substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 215; (c) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO: 215.

[0204] In certain embodiments, the composition further comprises:

[0205] (iv) a fourth component selected from (i) a pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof); (ii) a nucleotide sequence encoding the pseudouridine synthase; (iii) any combination of (i) and (ii);

[0206] The pseudouridine synthase (eg, DKC1 protein or a functional fragment thereof) can be guided or recruited by the engineered guide RNA and modify the target uridine residue in the target RNA hybridized with the engineered guide RNA into a pseudouridine residue.

[0207] In certain embodiments, the DKC1 protein or a functional fragment thereof comprises the sequence shown in SEQ ID NO:124.

[0208] Based on the contents disclosed in the present application, it is easy for those skilled in the art to understand that when the first to fourth components of the composition of the present application can each independently be a nucleotide sequence, therefore, in certain embodiments in which at least two of the first to fourth components are each a nucleotide sequence, the nucleotide sequences of the components can be respectively present in different nucleic acid molecules or vectors, or can be present in the same nucleic acid molecule or vector.

[0209] For example, in an embodiment where the first component is a nucleotide sequence encoding the first O-tRNA and the second component is a nucleotide sequence encoding the first O-tRNA, the nucleotide sequence of the first component and the nucleotide sequence of the second component may be present in different nucleic acid molecules or vectors, respectively, or may be present in the same nucleic acid molecule or vector.

[0210] Based on the contents disclosed in this application, those skilled in the art will readily understand that the various components in the composition provided in this application can be provided in any mixed or separate form.

[0211] In another aspect, the present application provides a vector system comprising one or more vectors, wherein the one or more vectors comprise:

[0212] (1) a first nucleotide sequence encoding a first O-tRNA, wherein the first O-tRNA is as defined above; optionally, the first nucleotide sequence is operably linked to a first regulatory element; and

[0213] (2) a second nucleotide sequence encoding a first O-RS, wherein the first O-RS is as defined above; optionally, the second nucleotide sequence is operably linked to a second regulatory element;

[0214] The first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors.

[0215] In certain embodiments, the one or more vectors further comprise:

[0216] (3) a third nucleotide sequence encoding an engineered guide RNA; the engineered guide RNA is as defined in claim 24 or 25; optionally, the third nucleotide sequence is operably linked to a third regulatory element.

[0217] In certain embodiments, the one or more vectors further comprise:

[0218] (4) a fourth nucleotide sequence encoding a pseudouridine synthase; the pseudouridine synthase is as defined in claim 26; optionally, the third nucleotide sequence is operably linked to a fourth regulatory element.

[0219] In certain embodiments, the first regulatory element is a promoter, such as an inducible promoter.

[0220] In certain embodiments, the second regulatory element is a promoter, such as an inducible promoter.

[0221] In certain embodiments, the third regulatory element is a promoter, such as an inducible promoter.

[0222] In certain embodiments, the fourth regulatory element is a promoter, such as an inducible promoter.

[0223] In certain embodiments, the one or more vectors further comprise: a nucleotide sequence encoding a second O-tRNA, and a nucleotide sequence encoding a second O-RS; wherein the second O-tRNA is selected from the tRNA mutants described above; and the second O-RS is capable of recognizing a second amino acid and aminoacylating the second O-tRNA with the second amino acid.

[0224] In certain embodiments, the second O-RS is capable of recognizing a second amino acid different from the first amino acid and aminoacylates the second O-tRNA with the second amino acid. In certain embodiments, the second O-tRNA recognizes a selector codon different from the selector codon recognized by the first O-tRNA.

[0225] In another aspect, the present application provides a delivery composition comprising a delivery vector and one or more selected from the following: the tRNA mutant, nucleic acid molecule, vector, composition, or carrier system described above.

[0226] In certain embodiments, the delivery composition comprises a composition as described above;

[0227] In certain embodiments, the delivery composition comprises a carrier system as described above.

[0228] In another aspect, the present application provides a kit comprising the tRNA mutant, nucleic acid molecule vector, host cell, composition, vector system, or delivery composition as described above.

[0229] In certain embodiments, the delivery composition comprises a composition as described above.

[0230] In certain embodiments, the delivery composition comprises a carrier system as described above.

[0231] In certain embodiments, the kit comprises a delivery composition as described above.

[0232] In certain embodiments, the kit further comprises a first amino acid as defined above.

[0233] In another aspect, the present application provides a translation system comprising:

[0234] (a) Component I, which is selected from: (i) a first O-tRNA, wherein the first O-tRNA is selected from the tRNA mutants described above; (ii) a nucleotide sequence encoding the first O-tRNA; (iii) any combination of (i) and (ii);

[0235] (b) a component II selected from: (i) a first O-RS, wherein the first O-RS is as defined above; (ii) a nucleotide sequence encoding the first O-RS; (iii) any combination of (i) and (ii);

[0236] as well as,

[0237] (c) Component III comprising a first amino acid as defined above.

[0238] In certain embodiments, the translation system comprises a host cell comprising the component I and the component II.

[0239] In certain embodiments, the translation system further comprises a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule) encoding a target protein, wherein the nucleic acid molecule comprises a selector codon (e.g., an amber codon, an ochre codon, or an opal codon) that is recognized by the first O-tRNA.

[0240] Based on the disclosure of this application, it is readily understood by those skilled in the art that the tRNA mutants provided herein can preferentially recognize Ψ (pseudouridine) modified codons (e.g., ΨGA, ΨAG, ΨAA), and thus can achieve insertion of a specific amino acid at a target position (e.g., a position containing a Ψ modified codon) in a position-specific manner. Therefore, in certain preferred embodiments, the selector codon of the nucleic acid molecule (e.g., RNA molecule) encoding the target protein is Ψ-modified (e.g., ΨGA, ΨAG, ΨAA).

[0241] In certain embodiments, the selector codon of the nucleic acid molecule encoding the target protein is not Ψ-modified. In such embodiments, the translation system preferably further comprises:

[0242] (d) component IV selected from: (i) an engineered guide RNA, wherein the engineered guide RNA is as defined above; (ii) a nucleotide sequence encoding the engineered guide RNA; (iii) any combination of (i) and (ii);

[0243] The guide sequence of the engineered guide RNA molecule is capable of hybridizing with the RNA molecule encoding the target protein.

[0244] Based on the disclosure of this application, it is easy for those skilled in the art to understand that the engineered guide RNA can guide or recruit the endogenous pseudouridine synthase (e.g., endogenous DKC1 protein) of the translation system, and can also guide or recruit the exogenous pseudouridine synthase (e.g., exogenous DKC1 protein or a functional fragment thereof) of the translation system.

[0245] In certain embodiments, the translation system contains an endogenous pseudouridine synthase (eg, an endogenous DKC1 protein).

[0246] In certain embodiments, the translation system does not contain an endogenous pseudouridine synthase (e.g., an endogenous DKC1 protein). In such embodiments, the translation system preferably further comprises:

[0247] (e) Component V, which is selected from: (i) pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof); (ii) a nucleotide sequence encoding the pseudouridine synthase; (iii) any combination of (i) and (ii);

[0248] The pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof) can be guided or recruited by the engineered guide RNA and modify the uridine residues (e.g., uridine residues in the selector codons, such as amber codons, ochre codons, or opal codons) in the RNA molecule encoding the target protein that hybridizes with the engineered guide RNA into pseudouridine residues.

[0249] In certain embodiments, the pseudouridine synthase is endogenous or exogenous to the host cell.

[0250] In certain embodiments, the translation system further comprises:

[0251] (i) a second O-tRNA, and / or a nucleotide sequence encoding the second O-tRNA; the second O-tRNA is selected from the tRNA mutants described above;

[0252] (ii) a second O-RS, and / or a nucleotide sequence encoding the second O-RS; wherein the second O-RS is capable of recognizing a second amino acid and aminoacylating the second O-tRNA with the second amino acid;

[0253] as well as,

[0254] (iii) the second amino acid that can be recognized by the second O-RS.

[0255] In certain embodiments, the second O-RS is capable of recognizing a second amino acid different from the first amino acid and aminoacylates the second O-tRNA with the second amino acid. In certain embodiments, the second O-tRNA recognizes a selector codon different from the selector codon recognized by the first O-tRNA.

[0256] In another aspect, the present application provides a method for producing a protein containing a target amino acid at a target position in a translation system, comprising:

[0257] (1) Providing: a translation system as described above;

[0258] (2) incorporating the target amino acid into the target position of the protein in response to the selector codon during translation of the protein, wherein the target position in the protein corresponds to the position of the selector codon in the nucleic acid molecule encoding the target protein, thereby producing a protein containing the target amino acid at the target position;

[0259] The target amino acid is the first amino acid recognized by the first O-tRNA.

[0260] In another aspect, the present application provides a method for producing a protein containing a target amino acid at a target position in a host cell, comprising:

[0261] (1) Providing a host cell, comprising:

[0262] (a) a first component selected from: (i) a first O-tRNA, wherein the first O-tRNA is selected from the tRNA mutants described above; (ii) a nucleotide sequence encoding the first O-tRNA; (iii) any combination of (i) and (ii);

[0263] (b) a second component selected from: (i) a first O-RS, wherein the first O-RS is as defined above; (ii) a nucleotide sequence encoding the first O-RS; (iii) any combination of (i) and (ii); and

[0264] (c) a third component comprising a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule) encoding a target protein, wherein the nucleic acid molecule comprises a selector codon (e.g., an amber codon, an ochre codon, or an opal codon) that is recognized by the first O-tRNA;

[0265] (2) culturing the host cell in the presence of a target amino acid; wherein the target amino acid is the first amino acid recognized by the first O-tRNA;

[0266] as well as,

[0267] (3) incorporating the target amino acid into the target position of the protein during translation of the protein, wherein the target position in the protein corresponds to the position of the selector codon in the nucleic acid molecule, thereby producing a protein containing the target amino acid at the target position.

[0268] In certain preferred embodiments, the selector codon of the nucleic acid molecule (eg, RNA molecule) encoding the target protein is Ψ-modified (eg, ΨGA, ΨAG, ΨAA).

[0269] In certain embodiments, the selector codon of the nucleic acid molecule encoding the target protein is not Ψ-modified. In such embodiments, the host cell preferably further comprises:

[0270] (d) a fourth component selected from: (i) an engineered guide RNA, wherein the engineered guide RNA is as defined above; (ii) a nucleotide sequence encoding the engineered guide RNA; (iii) any combination of (i) and (ii);

[0271] The guide sequence of the engineered guide RNA molecule is capable of hybridizing with the RNA molecule encoding the target protein.

[0272] Based on the disclosure of this application, it is easy for those skilled in the art to understand that the engineered guide RNA can guide or recruit the endogenous pseudouridine synthase of the host cell (e.g., endogenous DKC1 protein), and can also guide or recruit the exogenous pseudouridine synthase of the host cell (e.g., exogenous DKC1 protein or a functional fragment thereof).

[0273] In certain embodiments, the host cell contains an endogenous pseudouridine synthase (eg, an endogenous DKC1 protein).

[0274] In certain embodiments, the host cell does not contain endogenous pseudouridine synthase (e.g., endogenous DKC1 protein). In such embodiments, the host cell preferably further comprises:

[0275] (e) a fifth component selected from the group consisting of: (i) a pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof); (ii) a nucleotide sequence encoding the pseudouridine synthase; (iii) any combination of (i) and (ii);

[0276] The pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof) can be guided or recruited by the engineered guide RNA and modify the uridine residues (e.g., uridine residues in the selector codons, such as amber codons, ochre codons, or opal codons) in the RNA molecule encoding the target protein that hybridizes with the engineered guide RNA into pseudouridine residues.

[0277] In certain embodiments, the pseudouridine synthase is endogenous or exogenous to the host cell.

[0278] In certain embodiments, the host cell further comprises:

[0279] (A) a second O-tRNA, and / or a nucleotide sequence encoding the second O-tRNA; the second O-tRNA is selected from the tRNA mutants described above;

[0280] (B) a second O-RS, and / or a nucleotide sequence encoding the second O-RS; wherein the second O-RS is capable of recognizing a second amino acid and aminoacylating the second O-tRNA with the second amino acid;

[0281] as well as,

[0282] (C) the second amino acid that can be recognized by the second O-RS.

[0283] In certain embodiments, the second O-RS is capable of recognizing a second amino acid different from the first amino acid and aminoacylates the second O-tRNA with the second amino acid. In certain embodiments, the second O-tRNA recognizes a selector codon different from the selector codon recognized by the first O-tRNA.

[0284] In certain embodiments, the first component, the second component, the third component, the fourth component, and the fifth component are each independently derived from endogenous expression or exogenous delivery of the host cell.

[0285] In another aspect, the present application provides a tRNA mutant, nucleic acid molecule, vector, host cell, composition, vector system, or delivery composition, kit or translation system as described above for use in producing a protein containing a target amino acid at a target position.

[0286] Abbreviations

[0287] PTC premature termination codon, premature termination codon

[0288] SCR stop codon read-through

[0289] tRNA Pyl pyrrolysyl tRNA, pyrrolysine tRNA

[0290] PylRS pyrrolysyl-tRNA synthetase

[0291] GCE genetic code expansion

[0292] RCE RNA-modification-assisted codon expansion, RNA modification-assisted codon expansion

[0293] RRTS Ribosome Readthrough Score, ribosome readthrough score

[0294] ncAA non-canonical amino acid, non-natural amino acid

[0295] CbzK Nε-(benzyloxycarbonyl)-L-lysine, Nε-benzyloxycarbonyl-L-lysine

[0296] BocK Nε-(tert-butoxycarbonyl)-L-lysine, Nε-tert-butoxycarbonyl lysine

[0297] TetBu 3-(6-butyl-1,2,4,5-tetrazin-3-yl)-L-phenylalanine, 3-(6-butyl-1,2,4,5-tetrazin-3-yl)-L-phenylalanine

[0298] DKC1 Dyskeratosis Congenita 1, also known as NAP57 or Cbf5

[0299] gsnoRNA guide snoRNA, guide small nucleolar RNA

[0300] ASL anticodon stem-loop

[0301] Ψ is used herein to refer to pseudouracil or a nucleoside (pseudouridine) or nucleotide residue containing pseudouridine (pseudouridine acid residue).

[0302] DHU is used herein to refer to dihydrouracil or a nucleoside (dihydrouridine) or nucleotide residue containing dihydrouracil (dihydrouridylic acid residue).

[0303] I is used herein to refer to hypoxanthine or a nucleoside (inosine) or nucleotide residue containing hypoxanthine (inosinic acid residue, inosinic acid residue)

[0304] ΨGA contains a pseudouracil-modified stop codon UGA

[0305] ΨAA contains a pseudouracil-modified stop codon UAA

[0306] ΨAG contains the pseudouracil-modified stop codon UAG

[0307] Definition of terms

[0308] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0309] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0310] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0311] As used herein, the term "non-natural amino acid (ncAA)" refers to any other amino acid, modified amino acid, and / or amino acid analog that is not among the 20 natural amino acids. Non-natural amino acids can be either L-amino acids or D-amino acids.

[0312] As used herein, the term "aminoacyl-tRNA synthetase (aaRS or RS)" refers to an enzyme that specifically recognizes the side chain of a specific amino acid and a specific tRNA, catalyzing the aminoacylation reaction between the amino acid and the tRNA, thereby covalently linking the two. Aminoacyl-tRNA synthetases are key enzymes that accurately translate the genetic information of mRNA into the amino acid sequence of proteins.

[0313] As used herein, the term "orthogonal" refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and / or an orthogonal aminoacyl-tRNA synthetase (O-RS)) that functions with endogenous components of a cell or translation system at reduced efficiency or is unable to function with endogenous components of the cell compared to the corresponding molecule endogenous to the cell or translation system. With respect to tRNA and aminoacyl-tRNA synthetase, the term "orthogonal" refers to a tRNA that is unable to function with an endogenous tRNA synthetase or functions with reduced efficiency compared to an endogenous tRNA that functions with the endogenous tRNA synthetase; or an orthogonal aminoacyl-tRNA synthetase that is ineffective or functions with reduced efficiency compared to an endogenous tRNA synthetase that functions with the endogenous tRNA, such as less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency. An orthogonal molecule lacks a functional endogenous complementary molecule in the cell. In one embodiment, the orthogonal tRNA of the cell is aminoacylated by any endogenous RS at a reduced efficiency or is even 0 compared to the endogenous RS aminoacylation of the endogenous tRNA. In another embodiment, the orthogonal RS aminoacylates any endogenous tRNA in the cell of interest at a reduced efficiency or is even 0 compared to the endogenous RS aminoacylation of the endogenous tRNA. A second orthogonal molecule that can function together with the first orthogonal molecule can be introduced into the cell. For example, orthogonal tRNA / RS pairings include, for example, 45% efficiency, 50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90% efficiency, 95% efficiency, or 99% efficiency, or higher, of the introduced supplementary components or active orthogonal pairings, that function together in the cell and compared to a control (e.g., corresponding tRNA / RS endogenous pairing).

[0314] As used herein, an orthogonal aminoacyl-tRNA synthetase (O-RS) is an enzyme that preferentially aminoacylates an orthogonal tRNA (O-tRNA) with an amino acid within a cell or translation system of interest. The amino acid that the O-RS loads onto the O-tRNA can be any amino acid, whether natural, unnatural, or artificial.

[0315] As used herein, an orthogonal tRNA (O-tRNA) is a tRNA that is orthogonal to a cell or translation system of interest, wherein the tRNA is, for example: (1) identical or substantially similar to a naturally occurring tRNA; (2) derived from a naturally occurring tRNA by natural or artificial mutagenesis; (3) produced by any method that takes into account the wild-type or mutant tRNA sequence of (1) or (2); (4) homologous to a wild-type or mutant tRNA; (5) homologous to any exemplary tRNA that is a substrate for an orthogonal tRNA synthetase; or (6) a conservative variant of any exemplary tRNA that is a substrate for an orthogonal tRNA synthetase. The O-tRNA can be charged with an amino acid or in an uncharged state. In addition, it should be understood that the "O-tRNA" can optionally be charged (aminoacylated) with an unnatural amino acid by a related synthetase.

[0316] As used herein, the term "selector codon" refers to a codon that is recognized by O-tRNA during translation and is preferably not recognized by endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates the amino acid it carries, such as an unnatural amino acid, at this position in the polypeptide. Selector codons can include, for example, nonsense codons or stop codons (e.g., amber, ochre, and opal codons); four-base or more codons; rare codons; codons derived from natural or unnatural base pairs, etc. As used herein, the terms "nonsense codon" and "stop codon" have the same meaning and are used interchangeably, including UAA / TAA (ochre codons), UAG / TAG (amber codons), and UGA / TGA (opal codons).

[0317] It is easy to understand that since the base T in DNA corresponds to the base U in RNA, the terms "TAG codon" and "UAG codon" have the same meaning and can be used interchangeably, the terms "TAA codon" and "UAA codon" have the same meaning and can be used interchangeably, and the terms "TGA codon" and "UGA codon" have the same meaning and can be used interchangeably.

[0318] As used herein, the term "translation system" refers to the components that incorporate amino acids into a growing polypeptide chain (protein). Components of a translation system can include, for example, ribosomes, tRNAs, aminoacyl-tRNA synthetases, mRNAs, and the like. The O-tRNAs and / or O-RSs of the present invention can be incorporated into or part of an in vitro or in vivo translation system, for example, in non-eukaryotic cells, such as bacteria (e.g., E. coli), or in eukaryotic cells, such as yeast, mammalian cells, plant cells, algae cells, fungal cells, insect cells, and the like, or in a cell-free in vitro translation system.

[0319] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0320] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an initiation of replication site.

[0321] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including those derived from non-natural polypeptides as described herein.

[0322] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0323] Advantageous Effects of the Invention

[0324] Compared to the more powerful genetic code expansion technology GCE currently in the field, the tRNA decoder and the RCE system based on the tRNA decoder provided in this application have one or more of the following beneficial effects:

[0325] (1) The tRNA decoder of the present application can preferentially read through the ΨCodon (e.g., ΨGA,

[0326] ΨAA, ΨAG), thereby achieving selectivity for specific RNA transcripts;

[0327] (2) The tRNA decoder of this application has been verified to be specific across the entire translational genome and has minimal interference with non-target transcripts;

[0328] (3) When the tRNA decoder of the present application is used, while achieving efficient insertion of the amino acid at the target site, the off-target readthrough of the endogenous stop codon is very low;

[0329] (4) This tRNA decoder can be recognized by a variety of aminoacyl-tRNA synthetases, thereby achieving the insertion of a variety of amino acids.

[0330] It is suitable for various application scenarios such as protein function regulation and bioorthogonal reaction.

[0331] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0332] Figure 1 : Ribosome analysis showed that the GCE system caused significant stop codon readthrough in the entire translation group. Figure 1 Specifically, the RRTS (Ribosome Readthrough Score) of the GCE system and the control group under the ribosome profiling measurement method is shown.

[0333] Figure 2 : Readthrough effect of tRNA mutants on reporter genes containing UGA codons or ΨGA codons; wherein, the horizontal axis is the tRNA mutant name, and the vertical axis is the readthrough efficiency; "-" indicates no addition of unnatural amino acids; for AlvtRNA and mmtRNA, "+" indicates the addition of the unnatural amino acid CbzK (Nε-(benzyloxycarbonyl)-L-lysine), and for InttRNA, "+" indicates the addition of the unnatural amino acid BocK (Nε-Boc-L-lysine).

[0334] Figure 3 .mmtRNA Pyl The read-through effect of (UCA)-37G on a reporter gene containing UGA or ΨGA codons. (a) mmtRNA PylRepresentative fluorescence images of the LDLR reporter gene with UGA or ΨGA codons read through by (UCA)-37G in the presence (+CbzK) or absence of unnatural amino acids (-ncAA) in the system. (b) Quantification of GFP and mCherry fluorescence intensities in the fluorescence images and calculation of the readthrough efficiency (i.e., readthrough ratio) were performed. The ΨGA codon preference coefficient was then calculated by calculating the ratio of the readthrough efficiencies of ΨGA and UGA. (c) mmtRNA Pyl Representative fluorescence images of the (UCA)-37G readthrough of an AGXT reporter gene with either a UGA or ΨGA codon in the presence (+CbzK) or absence (-ncAA) of an unnatural amino acid in the system. (d) Quantitative readthrough efficiency and ΨGA codon preference coefficient; the reporter gene with a stop codon located in the LDLR sequence is designated LDLR-TGA, and the reporter gene with a stop codon located in the AGXT sequence is designated AGXT-TGA.

[0335] Figure 4 . Readthrough efficiency of tRNA mutants that prefer to read through the ΨAA codon on a reporter gene containing a UAA codon or a ΨAA codon; "-" indicates no addition of an unnatural amino acid; for AlvtRNA and mmtRNA, "+" indicates addition of the unnatural amino acid CbzK (Nε-(benzyloxycarbonyl)-L-lysine); for InttRNA, "+" indicates addition of the unnatural amino acid BocK (Nε-Boc-L-lysine).

[0336] Figure 5 . Readthrough efficiency of tRNA single-base mutants that prefer to read through the ΨAG codon on reporter genes containing UAG codons or ΨAG codons; "-" indicates no addition of unnatural amino acids; for AlvtRNA and mmtRNA, "+" indicates addition of the unnatural amino acid CbzK (Nε-(benzyloxycarbonyl)-L-lysine); for InttRNA, "+" indicates addition of the unnatural amino acid BocK (Nε-Boc-L-lysine).

[0337] Figure 6The readthrough efficiency of tRNA double base mutants that prefer ΨAG codons on reporter genes containing UAG codons or ΨAG codons; "-" indicates no unnatural amino acid is added; "+" indicates unnatural amino acid CbzK (Nε-(benzyloxycarbonyl)-L-lysine) is added for Alv tRNA and mmtRNA; "+" indicates unnatural amino acid BocK (Nε-Boc-L-lysine) is added for Int tRNA.

[0338] Figure 7 : tRNA decoders cause significantly less readthrough of stop codons across the whole proteome when achieving the same readthrough effect on target transcripts. Among them, Figure 7 a specifically shows the readthrough level of transcripts with UGA stop codon readthrough phenomenon in HEK293T transfected with GCE system, RCE system or control group under the ribosome profiling measurement method; Figure 7 b specifically shows the readthrough level of target transcripts in HEK293T transfected with GCE system or RCE system under the ribosome profiling measurement method. The readthrough level is represented by ribosome readthrough score (RRTS).

[0339] Figure 8 : tRNA decoders cause significantly less misinsertion of unnatural amino acids across the whole proteome when achieving the same readthrough effect on target transcripts. Figure 8 Specifically shows the comparison of off-target proteins in RCE and GCE systems and gene ontology enrichment analysis; wherein, (a) the Wayne diagram shows the correlation of off-target proteins induced by GCE and RCE systems. The numbers in the figure identify the protein species in each area. (b) Gene ontology (GO) enrichment analysis of off-target proteins shows that, compared with RCE system, GCE system leads to significantly enhanced enrichment characteristics of off-target proteins in related biological processes. The significance analysis is based on hypergeometric test (a probability test method based on hypergeometric distribution). The significance is represented by adjusted P value (p.adjust), and p.adjust value less than 0.01 indicates significant difference.

[0340] Sequence information

[0341] The description of the sequences involved in the present application is provided in the following table.

[0342] Table 1: Sequence information

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] Note: 1. In the nucleotide sequences of SEQ ID NOs: 148-169, 174-188, 191-209, and 212, "N" represents the anticodon "UCA," "UUA," or "CUA." In SEQ ID NO: 215, the guide sequence is represented by "(N m1 )","(N m2 )","(N m3 )","(N m4 )” indicates a nucleotide sequence consisting of m1, m2, m3, and m4 N, respectively, wherein each N is independently any one of A, U, G, or C; m1, m2, m3, and m4 are each independently an integer greater than 0, preferably, m1, m2, m3, and m4 are each independently 4, 5, 6, 7, 8, 9, 10, 11, or 12. In certain exemplary embodiments, the guide sequence “(N m1 )","(N m3 )" can be designed to target the upstream sequence of the stop codon, the guide sequence "(N m2 )","(N m4 )" can be designed to target the downstream sequence of the stop codon.

[0355] 2. It is easy for those skilled in the art to understand that in order to ensure the correct folding of tRNA and the stability of its structure, mature tRNA with biological activity usually has a high proportion of modified bases (e.g., T, Ψ, DHU and I and various methylated bases), and a "CCA" structure is connected to the 3' end. Such modifications are well known to those of ordinary skill in the art (for example, see Peschek J, Tuorto F. Interplay Between tRNA Modifications and Processing. J Mol Biol. 2025 Aug 15; 437(16): 169198. doi: 10.1016 / j.jmb.2025.169198. Epub 2025 May 22. PMID: 40404521.; the entire text of which is incorporated herein by reference). The sequences of each tRNA and its ASL shown in the table are intended to present the naked nucleotide sequence information of each tRNA and its ASL. Based on the contents disclosed in this application, it is easy for those skilled in the art to understand that each tRNA and tRNA mutant provided in this application not only includes its naked nucleotide sequence information, but also includes the various tRNA modifications contained therein as described above. DETAILED DESCRIPTION

[0356] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention). It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the present invention.

[0357] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0358] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0359] As mentioned above, based on extensive research, the inventors of this application developed a tRNA decoder that preferentially reads through ΨCodon on the basis of the reported wild-type pyrrolysine tRNA, thereby achieving the distinction between ΨCodon and unmodified stop codons, thereby realizing sequence-specific readthrough of stop codons.

[0360] A detailed description of wild-type pyrrolysine tRNA can be found in “A facile system for encoding unnatural amino acids in mammalian cells” (Chen et al, Angew. Chem. Int. Ed Engl. 2009), which is incorporated herein by reference in its entirety.

[0361] To more clearly describe the present invention, an exemplary experimental protocol for a tRNA decoder is provided below:

[0362] Example 1: GCE causes stop codon readthrough in the entire translation group

[0363] This example uses ribosome profiling technology to detect the effect of the GCE system (mmPylRS-306A384F / mmtRNAPyl and the unnatural amino acid CbzK) on the stop codon readthrough of all endogenous transcripts across the entire translationome.

[0364] Ribosome profiling technology is a molecular biology method based on deep sequencing. It uses the steric hindrance effect of ribosomes during the translation process to accurately capture the position of translating ribosomes across the entire genome, thereby systematically analyzing the dynamic changes in global protein translation in cells. The technical principle is: when mRNA undergoes an extension reaction in the ribosome, the extension complex will produce a spatial protection of about 30 nucleotides (nt) in length on the mRNA, forming a complex of ribosomes and mRNA that is resistant to nucleases. By optimizing the digestion conditions of ribonucleases, mRNA regions that are not protected by ribosomes can be specifically degraded, thereby retaining and enriching ribosome-protected fragments (RPFs) in the translation extension state. After deep sequencing, these characteristic fragments of approximately 28-32nt can reflect the precise occupancy information of ribosomes on mRNA with single-codon resolution. In recent years, this technology has been widely used in the global evaluation of tRNA decoding fidelity, the study of the mechanism of abnormal protein production caused by stop codon readthrough, and the molecular analysis of regulatory phenomena such as translational pausing (see Wang, J., Zhang, Y., Mendonca, CA et al. AAV-Delivered Suppressor tRNA Overcomes a NonsenseMutation in Mice. Nature 604, 343-348 (2022) and Wangen, JR & Green, R. Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides. eLife 9, e52611 (2020)).

[0365] To examine the direct expression of pyrrolysine tRNA (tRNAPyl ) and its synthetase on the stop codons of all transcripts, this example transfected HEK293T cells with GCE system and control. Specifically, HEK293T cells transfected with reporter gene plasmid were used as control group to indicate the stop codon readthrough event inside the cells; HEK293T cells transfected with GCE system (mmPylRS-306A384F / mmtRNA Pyl , and HEK293T cells expressing unnatural amino acids CbzK) were used as GCE treatment groups to evaluate the effects of wild-type mmtRNA on the expression of tRNAs without the introduction of sequence-specific design. Pyl (UCA) is a read-through event triggered by an endogenous stop codon. Specifically, the synthetase used in the transfected GCE (UGA) system is the reported chimeric synthetase mmPylRS-306A384F, and the tRNA is the wild-type tRNA Pyl The sequence is SEQ ID NO: 147. Transfection was performed using LTX plus reagent produced by Thermo and processed according to the manufacturer's standard procedures.

[0366] Subsequently, ribosome profiling libraries were constructed for cells transfected with the GCE system and with the DKC1-vector empty plasmid containing the CMV promoter or the promoter-less plasmid. The specific experimental procedures are as follows: (1) Cycloheximide treatment: Add fresh culture medium containing 0.1 mg / mL cycloheximide to the HEK293T cell culture dish and incubate for 1 minute. Discard the culture medium and wash twice with pre-cooled PBS containing 0.1 mg / mL cycloheximide. Collect the cells and centrifuge them. Quickly freeze them in liquid nitrogen and transfer them to dry ice. (2) Cell lysis and filtration: Add 400 μL of lysis buffer to each 10 cm culture dish, repeatedly pipette and lyse the cells 10 times, centrifuge at 20,000 × g for 10 minutes at 4°C, collect the supernatant, and filter it with a 0.2 μM filter membrane. (3) RNase I digestion: Add 7.5 μL RNase I (100 U / μL) to every 300 μL lysate, incubate with rotation at 25°C for 45 min, then add 10 μL SUPERase In to terminate the reaction and store on ice. (4) Purification of ribosome-protected fragments: Purify using a MicroSpin S-400 column, extract RNA with phenol-chloroform, separate the target fragments by TBE-urea gel electrophoresis, and recover RNA by gel excision. (5) rRNA removal and library construction: Remove rRNA using the Ribominus kit, and construct the library according to the standard protocol of the small RNA library construction kit. (6) Sequencing and data analysis: The raw sequencing data were trimmed to remove linkers and low-quality bases, and rRNA or tRNA alignment reads were filtered. The hg38 genome was aligned using bowtie (parameters: -m 1 -v 1 --best-strata). RiboWaltz was used to identify P sites, and the ribosome readthrough score (RRTS) was calculated. The Mann-Whitney U test was used to compare RRTS differences, and genes with a two-fold increase in RRTS > 0.02 were screened for GO enrichment analysis (clusterProfiler).Among them, RRTS is a method commonly used in the industry to evaluate the effect of tRNA on termination codon readthrough. For its specific evaluation method, please refer to Wang, J., Zhang, Y., Mendonca, CA et al. AAV-Delivered Suppressor tRNA Overcomes a Nonsense Mutation in Mice. Nature 604, 343-348 (2022); Wangen, JR & Green, R. Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides. eLife 9, e52611 (2020).

[0367] Ribosome analysis revealed that the GCE system caused an increase in the RRTS score of the 3'UTR region of gene transcripts in the whole translation group compared with the control group, indicating that it led to a significant stop codon read-through phenomenon ( Figure 1 ).

[0368] Example 2: Design and construction of tRNA decoder for Ψ codon

[0369] Currently, the most widely used aaRS-tRNA pair in the GCE system is MmPylRS / MmtRNA Pyl and the very similar MbPylRS / MbtRNA Pyl Yes. The reasons why these two unnatural aminoacyl-tRNA synthetase-tRNA pairs are popular are: (1) PylRS / tRNA Pyl The pair can function normally in both prokaryotic and eukaryotic cells and is orthogonal to the endogenous aminoacyl synthetase-tRNA pair in both prokaryotic and eukaryotic cells; (2) Since PylRS is sensitive to tRNA Pyl Recognition is independent of tRNA Pyl The anticodon region of tRNA Pyl The anticodon region of PylRS can be mutated to decode different codons without affecting its own aminoacylation; (3) the catalytic pocket of PylRS that recognizes amino acids can be modified to adapt to different types of ncAA substrates, thereby enabling the specific insertion of multiple ncAAs.

[0370] In order to obtain a tRNA decoder that can preferentially recognize Ψ-codon, the inventors of the present application have developed a novel tRNA decoder based on pyrrolysine tRNA (tRNA Pyl ) and synthetase binding characteristics, designed and constructed tRNAPyl Mutant library.

[0371] The inventors studied the naturally occurring tRNA Pyl Search and classification were performed, and MmtRNA was selected Pyl 、SpetRNA Pyl 、AlvtRNA Pyl and InttRNA Pyl These four tRNAs (derived from Methanosarcina mazei, Methanosarcinaspelaei Spe, Methanomethylophilus alvus, and Methanomassiliicoccus intestinalis, respectively) were used as the basic framework for modification. After determining the basic framework, the inventors analyzed and explored the regions of tRNA that could be modified based on the crystal structure of the tRNA and synthetase complex, and selected the tRNA anticodon stem-loop region (positions 27-43) for single-base mutation to obtain tRNA Pyl Mutation library.

[0372] Example 3: Construction of RCE system and evaluation of read-through efficiency based on dual-color fluorescent protein reporter system

[0373] In order to achieve sequence-specific stop codon recognition and read-through, sequence-specific guide RNA (guidesnoRNA, gsnoRNA) is used to guide pseudouracil synthetase to carry out Ψ modification tag on the target sequence, so that the tRNA that prefers to identify Ψ modification stop codon (Ψ-Codon) can specifically identify the Ψ modification stop codon. In this application, this specific stop codon is subjected to pseudouracil modification to produce Ψ Codon, and then the method for specifically identifying Ψ Codon with tRNA decoder is referred to as RNA modification-assisted codon expansion (RCE) technology. To achieve Ψ modification in the composite system of aaRS and its corresponding tRNA of co-transfection ncAA, an exemplary scheme of the RCE technology provided in this application adopts a quaternary co-transfection strategy, in which gsnoRNA, reporter gene vector, ncAA aminoacyl-tRNA synthetase expression vector and tRNA expression vector are synchronously transfected into host cells.

[0374] In order to evaluate the readthrough efficiency of the transfected RCE system on the stop codon of the target gene, this embodiment constructed a dual-color fluorescent reporter gene system containing a stop codon. The reporter gene consists of the coding sequence of the red fluorescent protein mCherry and the green fluorescent protein GFP, which are connected in series through a linker sequence containing the target stop codon to construct a reporter gene for detecting the readthrough efficiency of tRNA. Since there is an early stop codon between the red fluorescent protein (mCherry) and the green fluorescent protein (GFP) in the reporter gene plasmid for evaluating the readthrough effect, when no readthrough event occurs, there is only red fluorescence but no green fluorescence, so the ratio of green fluorescence intensity to red fluorescence intensity can be used as a quantitative readthrough effect.

[0375] When the reporter gene is successfully transfected into the cells, the fluorescence intensity of the red fluorescence of mCherry is used as the internal reference, and the ratio of the green fluorescence intensity of GFP to the red fluorescence intensity of mCherry is used as the indicator of read-through efficiency.

[0376] Taking the evaluation of tRNA readthrough efficiency at UGA and ΨGA as an example, the specific experimental method is as follows:

[0377] Cell seeding: HEK293T cells were washed with PBS and treated with 0.25% trypsin at 37°C for 1 minute. Trypsin activity was terminated by adding FBS-containing medium. Cells were centrifuged at 500 × g for 5 minutes, counted, and diluted to 4 × 10 5 Subsequently, the cells were seeded into 96-well flat-bottom plates (LABSELECT, 11514) that had been pre-coated overnight with 20 ng / μL poly-D-lysine (Beyotime, ST508) solution and rinsed with water, and incubated for 16-24 hours.

[0378] Transfection: During transfection, the synthetase for MmtRNA was the aminoacyl-tRNA synthetase mmPylRS-306A384F (CbzK) (SEQ ID NO: 139), which recognizes the unnatural amino acid CbzK; the synthetase for AlvtRNA was the aminoacyl-tRNA synthetase 1R26PylRS (CbzK) (SEQ ID NO: 140), which recognizes the unnatural amino acid CbzK; and the synthetase for InttRNA was the aminoacyl-tRNA synthetase RumEnPylRS (BocK) (SEQ ID NO: 141), which recognizes the unnatural amino acid BocK.

[0379] For each tRNA mutant, a corresponding "ΨGA" mixture and "UGA" mixture were prepared. The "ΨGA" mixture was prepared by mixing 62.5 ng of Screen-TGA reporter vector (core region sequence of SEQ ID NO: 52), 15.6 ng of gsno-Screen (SEQ ID NO: 54), 7.8 ng of DKC1-iso3 (DKC1 splice isomer 3, SEQ ID NO: 124), 62.5 ng of synthetase (SEQ ID NO: 139), and 122.5 ng of the tRNA mutant with 0.5 μL of Lipofectamine LTX reagent (Invitrogen) and 0.2 μL of PLUS reagent (Invitrogen), following the manufacturer's instructions. Similarly, the "UGA" mixture was prepared by mixing 62.5 ng of the Screen-TGA reporter vector, 15.6 ng of gggctrl (non-targeting empty vector, SEQ ID NO: 126), 7.8 ng of the DKC1-vector, 62.5 ng of the synthetase aminoacyl-tRNA synthetase mmPylRS-306A384F (CbzK) (SEQ ID NO: 139), and 122.5 ng of the tRNA mutant with 0.5 μL of Lipofectamine LTX reagent and 0.2 μL of PLUS reagent in the same manner. The prepared "UGA" and "ΨGA" mixtures were added to wells seeded with HEK293T cells, and the culture medium was supplemented with 200 μM of the unnatural amino acid.

[0380] Fluorescence detection and read-through efficiency calculation: 48-72 hours after transfection, use Cell imaging was performed using a Micro 4 High-Content Imaging System (Molecular Devices LLC, Sunnyvale, CA). Images of four different areas in a single well of a 96-well plate were captured using a 10x objective and automatically analyzed using MetaXpress software (MX version 6.2.3.733; CME version 6.2.3.991). The intensity of mCherry or EGFP in fluorescence images was calculated by multiplying the single-cell fluorescence intensity by the number of positive cells. Readthrough efficiency was calculated as the ratio of GFP intensity to mCherry intensity and was normalized to the untruncated dual-color fluorescent reporter gene.

[0381] The preference of each tRNA for ΨGA was evaluated by comparing the readthrough efficiency of each tRNA under "UGA" and "ΨGA" conditions.

[0382] Correspondingly, when evaluating tRNA readthrough efficiency under UAA and ΨAA conditions, the above experimental procedures remain unchanged, except that the Screen-TGA reporter gene is replaced by the Screen-TAA reporter gene. Similarly, when evaluating tRNA readthrough efficiency under UAG and ΨAG conditions, the above experimental procedures remain unchanged, except that the Screen-TGA reporter gene is replaced by the Screen-TAG reporter gene.

[0383] Example 4: tRNA mutants that prefer the ΨGA codon

[0384] Based on four wild-type tRNA Pyl A library of single-base tRNA mutants with the UCA anticodon was constructed (SEQ ID NOs: 145-147, 66). Using the Screen-TGA dual-color fluorescent reporter gene containing the UGA stop codon, a high-content fluorescence screening system was used to evaluate the readthrough effect of each tRNA mutant on reporter genes containing the UGA codon and the ΨGA codon.

[0385] This method was used to screen tRNA mutants with ΨGA codon preference, and their sequences are shown in SEQ ID NOs: 1-10, 64-65. The readthrough effect of these tRNA mutants on the ΨGA codon was significantly higher than that on the UGA codon ( Figure 2 ).

[0386] Among them, mmtRNAPyl(UCA)-37G (SEQ ID NO: 8) clearly prefers to read through the ΨGA codon, and the read-through effect of this tRNA decoder on the ΨCodon is 2.6-9 times that of the unmodified codon ( Figure 3 The tRNA mutants that showed a preference for the ΨGA codon obtained by screening were called tRNA decoders for the ΨGA codon.

[0387] Example 5: tRNA mutants that prefer ΨAA codons

[0388] This example is based on wild-type tRNA Pyl(SEQ ID NO: 142-144) A library of tRNA mutants that preferentially recognize ΨAA codons rather than UAA codons was constructed. Subsequently, a Screen-TAA dual-color fluorescent reporter gene containing a UAA stop codon was used to evaluate the read-through effect of each tRNA mutant on a reporter gene containing a UAA codon and a reporter gene containing a ΨAA codon using a high-content fluorescent screening system. The evaluation method is shown in Example 3. The tRNA mutants that prefer to read through ΨAA codons rather than UAA codons were screened, and their sequences are shown in SEQ ID NO: 11 to 15. The read-through effect is shown in Figure 4 Because these tRNA decoders show a readthrough preference for the ΨAA codon, they are referred to as tRNA decoders for the ΨAA codon.

[0389] Example 6: tRNA single-base mutants that prefer the ΨAG codon

[0390] Based on wild-type tRNA Pyl (SEQ ID NO: 68-70) A library of tRNA mutants that preferentially recognize ΨAG codons rather than UAG codons was constructed. Subsequently, using the Screen-TAG dual-color fluorescent reporter gene containing the UAG codon, based on a high-content fluorescence screening system, the readthrough effect of each tRNA mutant on the reporter gene containing the UAG codon and the reporter gene containing the ΨAG codon was evaluated. tRNA mutants that preferentially read through the ΨAG codon rather than the UAG codon were screened ( Figure 5 ), whose sequences are shown in SEQ ID NOs: 16 to 50. Since these tRNA decoders show a readthrough preference for the ΨAG codon, they are referred to as tRNA decoders for the ΨAG codon.

[0391] Example 7: tRNA double base mutants preferring the ΨAG codon

[0392] In order to further improve the read-through effect and specificity of the tRNA decoder of the ΨAG codon, the present application carried out a combined double-base mutation based on the results of the single-base mutation library to further improve the specificity of the tRNA decoder. Using the Screen-TAG dual-color fluorescent reporter gene containing the UAG stop codon, a high-content fluorescence screening system was used to evaluate the read-through effect of each tRNA mutant on the reporter gene containing the UAG codon and the reporter gene containing the ΨAG codon. The tRNA double-base mutant ( Figure 6 ), whose sequences are shown in SEQ ID NOs: 51, 53, 55-63.

[0393] Example 8: The RCE system achieves sequence-specific stop codon readthrough across the entire translational genome

[0394] In this example, the mmtRNA obtained by screening Pyl Using the (UCA)-37G mutant as an example, the stop codon recognition and read-through specificity of the RCE system using this mutant as a tRNA decoder in the entire translation group were verified. In this example, the read-through events of the stop codons were compared in HEK293T cells that were transfected with a reporter gene plasmid, a GCE system, or an RCE system (including gsnoRNA and tRNA decoders for coding). Among them, HEK293T cells transfected with a reporter gene plasmid were used as a control group to indicate the stop codon read-through events inside the cells. The commonly used GCE system (mmPylRS-306A384F / mmtRNA Pyl , and HEK293T cells expressing unnatural amino acids CbzK) were used as GCE treatment groups to evaluate the effects of wild-type mmtRNA on the expression of tRNAs without the introduction of sequence-specific design. Pyl (UCA) triggers a read-through event at the endogenous stop codon. Pyl (UCA)-37G (SEQ ID NO: 8) cells were used as the RCE system treatment group to evaluate whether the RCE system can exhibit sequence specificity after the introduction of the encoding and decoding modules, thereby ensuring the translation efficiency of the target sequence while reducing the read-through interference of the endogenous stop codon.

[0395] The ribosome profiling technique described in Example 1 was used to detect the RRTS scores of the 3'UTR region of gene transcripts in the whole translation group. The results showed that the median values ​​of RRTS in the GCE system, RCE system, and control cells were 0.057, 0.014, and 0. Figure 7 a) The stop codon readthrough level on endogenous transcripts in cells transfected with the RCE system is approximately 1 / 4 of that in the GCE system, indicating that the RCE system reduces the global off-target readthrough rate by approximately 4-fold.

[0396] Furthermore, sequencing analysis showed that the read-through efficiency of the RCE system on the target transcript was similar to that of GCE cells ( Figure 7 b). Therefore, given similar readthrough efficiencies across the target sequence for both the GCE and RCE systems, the reduction in global off-target readthrough suggests that the RCE system exhibits approximately a four-fold improvement in specificity for the target transcript across the entire translatome. Together, these results suggest that while the tRNA decoder achieves the same readthrough efficiency across the target transcript, it induces significantly less stop codon readthrough across the entire translatome.

[0397] Example 9: RCE system achieves sequence-specific unnatural amino acid insertion across the entire proteome

[0398] In this example, MmtRNA was used Tet (UCA)-37G (SEQ ID NO: 65) was used as the tRNA decoder in the RCE system, and proteomic analysis technology was used to evaluate the unnatural amino acid mis-insertion events caused by the RCE system in non-target proteins across the entire proteome.

[0399] The tRNATet decoder can introduce the chemically bond-forming, unnatural amino acid TetBu (3-(6-butyl-1,2,4,5-tetrazin-3-yl)-L-phenylalanine) into target proteins. TetBu reacts with TCO-biotin, introducing a biotin tag into the TetBu-incorporated protein. This biotin-tagged protein can then be captured using streptavidin magnetic beads, enabling specific enrichment of TetBu-expressing proteins.

[0400] The specific experimental methods are as follows:

[0401] In order to compare the off-target insertion of ncAA in the GCE system and the RCE system, this study evaluated the off-target insertion of TetBu in endogenous proteins by the two methods using protein analysis. The components of the GCE system are TetBuRS (SEQ ID NO: 67), DKC1-vector, MmtRNA Tet (UCA) (SEQ ID NO: 72) and gctrl; the components of the RCE system are TetBuRS (SEQ ID NO: 67), DKC1-iso3 (DKC1 3 splice isomer, SEQ ID NO: 124), MmtRNA Tet (UCA)-37G (SEQ ID NO: 65) and gsnoRNA-Screen (SEQ ID NO: 54); control cells were transfected with CMV promoter-containing and promoter-less DKC1-vector empty plasmids.

[0402] 48 hours after transfection, HEK293T cells were harvested with ice-cold PBS and centrifuged at 500 × g for 3 minutes. The supernatant was discarded and the cells were lysed in RIPA buffer at 37°C for 10 minutes. Three volumes of 8M Urea / PBS were added and incubated at 37°C for 10 minutes. After centrifugation, the supernatant was removed and reacted with 50 μM TCO-biotin at 37°C for 2 hours. Subsequently, 100 μM TetBu was added for 10 minutes. The lysate was precipitated with methanol-chloroform, frozen at -30°C for 1 hour, centrifuged at 12,000 rpm for 10 minutes, the supernatant discarded, and the cells were washed twice with anhydrous methanol. For protein mass spectrometry identification, the cells were reconstituted in 2% SDS / PBS, sonicated, and diluted to 0.2% SDS / PBS. The cells were then incubated with streptavidin magnetic beads for 3 hours to capture TetBu-incorporated proteins. (Since TetBu and TCO-biotin react, the TetBu-incorporated proteins will have a biotin signal that can be captured by the streptavidin magnetic beads.) After dithiothreitol reduction and iodoacetamide alkylation, it was dissolved in 2M urea PBS and digested with trypsin at 37°C for 16 hours. The enzymatic cleavage product was desalted and dissolved in 0.1% formic acid for LC-MS / MS analysis (timsTOF Pro2), and the data was processed by Maxquant. Subsequently, the control group was used as the background of the endogenous biotin sample, and the specific types of off-target proteins were obtained by identifying the peptides. After performing type analysis and abundance analysis on the captured biotinylated proteins based on LC-MS / MS, this example compared the protein abundance between different samples.

[0403] Analysis of the types of TetBu-inserted proteins detected in the RCE and GCE groups using proteomic analysis revealed that when the tRNA decoder achieves the same read-through effect on the target transcript, the number of proteins with unnatural amino acid misinsertion caused by it in the whole proteome is significantly less ( Figure 8 a), specifically: 113 proteins were detected in the proteomic data of cells transfected with GCE; while only 44 proteins were detected in the proteomic data of cells transfected with RCE. In addition, GO (Gene Ontology) analysis of proteins detected in the GCE and RCE groups revealed that the biological pathways affected by RCE were far fewer than those of the GCE system. The off-target proteins in the GCE system were enriched in 13 pathways, while the off-target proteins in the RCE system were only weakly enriched in 5 pathways ( Figure 8 b), thus proving that the decoding accuracy of the tRNA decoder of the present application is higher.

[0404] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A tRNA mutant comprising a nucleotide substitution mutation at one or more positions selected from the group consisting of: Position 27, position 28, position 29a, position 29b, position 30, position 31, position 32, position 33, position 37, position 38, position 39, position 40, position 41a, position 41b, position 41c, position 42, position 43.

2. The tRNA mutant of claim 1, comprising one or more mutations selected from the group consisting of: (1) 27G; (2) 28G or 28U; (3) 29aA or 29aU; (4) 29bA or 29bU; (5) 30U; (6) 31C, 31A, 31G, or 31U; (7) 32U or 32A; (8) 33A or 33C; (9) 37A, 37G, 37U, or 37C; (10) 38U or 38C; (11) 39A or 39C; (12) 40U; (13) 41aU or 41aG; (14) 41bC; (15) 41cA or 41cG; (16) 42A or 42C; (17) 43A, 42C, or 43U; Preferably, compared to the wild-type tRNA, the tRNA mutant comprises one or more mutations selected from the group consisting of: (1) 31C, 31A, 31G or 31U; (2) 33A or 33C; (3) 37A, 37G, 37U or 37C; (4) 39A or 39C.

3. The tRNA mutant according to claim 1 or 2, comprising a mutation selected from the group consisting of: (1) 27G; (2) 28G or 28U; (3) 29aA or 29aU; (4) 29bA or 29bU; (5) 30U; (6) 31C, 31A, 31G, or 31U; (7) 32U or 32A; (8) 33A or 33C; (9) 37A, 37G, 37U, or 37C; (10) 38U or 38C; (11) 39A or 39C; (12) 40U; (13) 41aU or 41aG; (14) 41bC; (15) 41cA or 41cG; (16) 42A or 42C; (17) 43A, 42C or 43U; (18) 31C and 37C; (19) 31G and 37C; (20) 31U and 37C; (21) 30U and 37G; (22) 31C and 33C; (23) 31C and 37G; (24) 31G and 33C; (25) 31G and 37G; (26) 31U and 33C; (27) 31U and 37G; (28) 33C and 37G; Preferably, the tRNA mutant comprises a mutation selected from the group consisting of: (1) 29aA or 29aU; (2) 29bA or 29bU; (3) 31C, 31A, 31G or 31U; (4) 33A or 33C; (5) 37A, 37G, 37U or 37C; (6) 39A or 39C; (7) 31C and 37C; (8) 31G and 37C; (9) 31U and 37C; (10) 31C and 33C; (11) 31C and 37G; (12) 31G and 33C; (13) 31G and 37G; (14) 31U and 33C; (15) 31U and 37G; (16) 33C and 37G.

4. The tRNA mutant according to any one of claims 1 to 3, wherein The wild-type tRNA is selected from: tRNA derived from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), tRNAs derived from Methanosarcina spelaei Spe (Methanosarcina spelaei Spe) (e.g., SpetRNA Pyl ), tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), tRNAs derived from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ).

5. The tRNA mutant according to any one of claims 1 to 4, which is Pyl 、MmtRNA Tet ), comprising one or more mutations selected from the group consisting of: (1) 30U; (2) 31C, 31G or 31U; (3) 33A or 33C; (4) 37G or 37C; (5) 38U or 38C; (6) 39A or 39C; (7) 40U; Preferably, compared to the wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of: (1) 30U; (2) 31C, 31G or 31U; (3) 33A or 33C; (4) 37G or 37C; (5) 38U or 38C; (6) 39A or 39C; (7) 40U; (8) 31C and 37C; (9) 31G and 37C; (10) 31U and 37C; (11) 30U and 37G; (12) 31C and 33C; (13) 31C and 37G; (14) 31G and 33C; (15) 31G and 37G; (16) 31U and 33C; (17) 31U and 37G; (18) 33C and 37G; Preferably, compared to the wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of: (1) 31C, 31G, or 31U; (2) 33A or 33C; (3) 37G or 37C; (4) 39A or 39C; (5) 31C and 37C; (6) 31G and 37C; (7) 31U and 37C; (8) 31C and 33C; (9) 31C and 37G; (10) 31G and 33C; (11) 31G and 37G; (12) 31U and 33C; (13) 31U and 37G; (14) 33C and 37G; Preferably, the tRNA mutant comprises an ASL (anticodonstem-loop) sequence selected from any one of SEQ ID NOs: 148-169; Preferably, the tRNA mutant comprises the sequence shown below: S1-S ASL -S2 in, S1 comprises a sequence as shown in SEQ ID NO: 170 or 172 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 148-169; S2 comprises a sequence as shown in SEQ ID NOs: 171 or 173 or a variant thereof; The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

6. The tRNA mutant of claim 5, comprising the anticodon CUA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 30U; (ii) 31C, 31G or 31U; (iii) 33A or 33C; (iv) 37G or 37C; (v) 38U or 38C; (v) 40U; preferably, compared to the wild-type tRNA derived from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of: (i) 30U; (ii) 31C, 31G, or 31U; (iii) 33A or 33C; (iv) 37G or 37C; (v) 38U or 38C; (vi) 40U; (vii) 31C and 37C; (viii) 31G and 37C; (ix) 31U and 37C; (x) 30U and 37G; (xi) 31C and 33C; (xii) 31C and 37G; (xiii) 31G and 33C; (xiv) 31G and 37G; (xv) 31U and 33C; (xvi) 31U and 37G; (xvii) 33C and 37G; preferably, compared to a wild-type tRNA derived from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises a mutation selected from the group consisting of: (i) 31C, 31G, or 31U; (ii) 33A or 33C; (iii) 37G or 37C; (iv) 31C and 37C; (v) 31G and 37C; (vi) 31U and 37C; (vii) 31C and 33C; (viii) 31C and 37G; (ix) 31G and 33C; (x) 31G and 37G; (xi) 31U and 33C; (xii) 31U and 37G; (xiii) 33C and 37G; (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 116-123, 125, and 127-136; (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 44-51, 53, 55-63 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

7. The tRNA mutant of claim 5, comprising an anticodon UCA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 31C; (ii) 37G; (iii) 39A or 39C; (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 79-82 and 138; (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 7-10, 65, or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

8. The tRNA mutant of claim 5, comprising an anticodon UUA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanosarcina mazei (e.g., MmtRNA Pyl 、MmtRNA Tet ), the tRNA mutant comprises the mutation: 37G; (2) the tRNA mutant comprises an ASL sequence selected from the group consisting of SEQ ID NO: 87; (3) The tRNA mutant comprises a sequence selected from SEQ ID NO: 15 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

9. The tRNA mutant according to any one of claims 1 to 4, which is different from the wild-type tRNA derived from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), comprising one or more mutations selected from the group consisting of: (1) 28G or 28U; (2) 29aA or 29aU; (3) 29bA or 29bU; (4) 31C or 31A; (5) 32U or 32A; (6) 33A or 33C; (7) 37C; (8) 38U; (9) 41aU or 41aG; Preferably, compared to the wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), the tRNA mutant comprises a mutation selected from the group consisting of: (1) 28G or 28U; (2) 29aA or 29aU; (3) 29bA or 29bU; (4) 37C; Preferably, the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 174-188; Preferably, the tRNA mutant comprises the sequence shown below: S1-S ASL -S2 in, S1 comprises the sequence shown in SEQ ID NO: 189 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 174-188; S2 comprises a sequence as shown in SEQ ID NO: 190 or a variant thereof; The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

10. The tRNA mutant of claim 9, comprising the anticodon CUA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 28G or 28U; (ii) 29aU; (iii) 29bA or 29bU; (iv) 31C or 31A; (v) 32A; (vi) 37C; (vii) 38U; (viii) 41aG; preferably, compared to the wild-type tRNA derived from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), wherein the tRNA mutant comprises a mutation selected from the group consisting of: (i) 28G or 28U; (ii) 29aU; (iii) 29bA or 29bU; (iv) 37C; (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 88-98; (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 16-26 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

11. The tRNA mutant of claim 9, comprising an anticodon UCA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 32U; (ii) 33A or 33C; (iii) 41aU; (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 73-76; (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 1-4 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

12. The tRNA mutant of claim 9, comprising an anticodon UUA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanomethylophilus alvus (e.g., AlvtRNA Pyl ), the tRNA mutant comprises the mutation: 29aA; (2) the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 83; (3) The tRNA mutant comprises a sequence selected from SEQ ID NO: 11 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

13. The tRNA mutant according to any one of claims 1 to 4, which is Pyl ), comprising one or more mutations selected from the group consisting of: (1) 27G; (2) 28U; (3) 29aU; (4) 31U; (5) 33C; (6) 37A, 37G, or 37U; (7) 41aU; (8) 41bC; (9) 41cA or 41cG; (10) 42A or 42C; (11) 43A, 42C, or 43U; Preferably, compared to the wild-type tRNA derived from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises a mutation selected from the group consisting of: (1) 28U; (2) 37A, 37G, or 37U; (3) 41aU; (4) 41bC; (5) 41cA or 41cG; Preferably, the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 191-209; Preferably, the tRNA mutant comprises the sequence shown below: S1-S ASL -S2 in, S1 comprises the sequence shown in SEQ ID NO: 210 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from any one of SEQ ID NOs: 191-209; S2 comprises a sequence as shown in SEQ ID NO: 211 or a variant thereof; The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

14. The tRNA mutant of claim 13, comprising the anticodon CUA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), wherein the tRNA mutant comprises one or more mutations selected from the group consisting of: (i) 27G; (ii) 28U; (iii) 29aU; (iv) 31U; (v) 33C; (vi) 37A, 37G, or 37U; (vii) 41aU; (viii) 41bC; (ix) 41cA or 41cG; (x) 42A or 42C; (xi) 43A, 43C, or 43U; preferably, compared to the wild-type tRNA derived from Methanomas siliicoccus intestinalis (e.g., InttRNA Pyl ), wherein the tRNA mutant comprises a mutation selected from the group consisting of: (1) 28U; (2) 37G or 37U; (3) 41aU; (4) 41bC; (5) 41cA or 41cG; (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 99-115; (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 27-43 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

15. The tRNA mutant of claim 13, comprising an anticodon UCA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises mutation: 37A or 37G; (2) the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 77 or 78; (3) The tRNA mutant comprises a sequence selected from SEQ ID NO: 5 or 6 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

16. The tRNA mutant of claim 13, comprising an anticodon UUA; Preferably, the tRNA mutant has one or more characteristics selected from the following: (1) Compared with wild-type tRNA from Methanomassiliicoccus intestinalis (e.g., InttRNA Pyl ), the tRNA mutant comprises the mutation: 37A, 37G or 37U; (2) the tRNA mutant comprises an ASL sequence selected from any one of SEQ ID NOs: 84-86; (3) The tRNA mutant comprises a sequence selected from any one of SEQ ID NOs: 12-14 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

17. The tRNA mutant according to any one of claims 1 to 4, which is Pyl ), containing mutations: 37G; Preferably, the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 212; Preferably, the tRNA mutant comprises the sequence shown below: S1-S ASL -S2 in, S1 comprises the sequence shown in SEQ ID NO: 213 or a variant thereof; ASL is an ASL sequence comprising a sequence selected from the group consisting of SEQ ID NO: 212; S2 comprises a sequence as shown in SEQ ID NO: 214 or a variant thereof; The variant has one or more nucleotide substitutions, deletions or additions compared to the sequence from which it is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions, deletions or additions).

18. The tRNA mutant of claim 17, comprising an anticodon UCA; Preferably, the tRNA mutant comprises an ASL sequence selected from SEQ ID NO: 137; Preferably, the tRNA mutant comprises a sequence selected from SEQ ID NO: 64 or a variant thereof; wherein, The variant has a substitution, deletion or addition of one or several nucleotides compared to the sequence from which it is derived (e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).

19. A nucleic acid molecule encoding the tRNA mutant according to any one of claims 1 to 18.

20. A vector comprising the nucleic acid molecule of claim 19.

21. A host cell comprising the nucleic acid molecule of claim 19 or the vector of claim 20.

22. A composition comprising: (a) a first component selected from: (i) a first orthogonal tRNA (O-tRNA), wherein the first O-tRNA is selected from the tRNA mutant of any one of claims 1 to 18; (ii) a nucleotide sequence encoding the first O-tRNA; (iii) any combination of (i) and (ii); as well as, (b) a second component selected from: (i) a first orthogonal aminoacyl-tRNA synthetase (O-RS) that recognizes a first amino acid and aminoacylates the first O-tRNA with the first amino acid; (ii) a nucleotide sequence encoding the first O-RS; or (iii) any combination of (i) and (ii).

23. The composition of claim 22, wherein The first amino acid is selected from natural amino acids and unnatural amino acids (e.g., Nε-benzyloxycarbonyl-L-lysine (CbzK), Nε-(cis-cyclooct-2-ene-1-oxycarbonyl)-L-lysine (TCOK), 3-(6-butyl-1,2,4,5-tetrazin-3-yl)-L-phenylalanine (TetBu), Nε-tert-butyloxycarbonyl-L-lysine (BocK)).

24. The composition of claim 22 or 23, further comprising: (iii) a third component selected from (i) an engineered guide RNA; (ii) a nucleotide sequence encoding the engineered guide RNA; (iii) any combination of (i) and (ii); in, The engineered guide RNA comprises a backbone sequence and a guide sequence, wherein the guide sequence is capable of hybridizing with a target RNA, and the guide RNA is capable of guiding or recruiting a pseudouridine synthase (e.g., a DKC1 protein) to modify a target uridine residue in the target RNA hybridized with the engineered guide RNA into a pseudouridine residue.

25. The composition of claim 24, wherein The engineered guide RNA is an engineered guide small nucleolar RNA (gsnoRNA); Preferably, the backbone sequence of the engineered gsnoRNA is derived from wild-type H / ACA-snoRNA: ACA2b, ACA36, ACA44, ACA27, E2, ACA3, and ACA17; Preferably, the sequence of the engineered gsnoRNA is selected from: (a) a sequence as shown in SEQ ID NO: 215; (b) a sequence having one or more nucleotide residue substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide residue substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 215; (c) a sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO:

215.

26. The composition of claim 24 or 25, further comprising: (iv) a fourth component selected from (i) a pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof); (ii) a nucleotide sequence encoding the pseudouridine synthase; (iii) any combination of (i) and (ii); in, The pseudouridine synthase (eg, DKC1 protein or a functional fragment thereof) can be guided or recruited by the engineered guide RNA and modify the target uridine residue in the target RNA hybridized with the engineered guide RNA into a pseudouridine residue.

27. A vector system comprising one or more vectors, said one or more vectors comprising: (1) a first nucleotide sequence encoding a first O-tRNA, wherein the first O-tRNA is as defined in claim 22 or 23; optionally, the first nucleotide sequence is operably linked to a first regulatory element; and (2) a second nucleotide sequence encoding a first O-RS, wherein the first O-RS is as defined in claim 22 or 23; optionally, the second nucleotide sequence is operably linked to a second regulatory element; in, The first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors.

28. The vector system of claim 27, wherein The one or more vectors further comprise: (3) a third nucleotide sequence encoding an engineered guide RNA; the engineered guide RNA is as defined in claim 24 or 25; optionally, the third nucleotide sequence is operably linked to a third regulatory element.

29. The vector system of claim 28, wherein The one or more vectors further comprise: (4) A fourth nucleotide sequence encoding a pseudouridine synthetase; the pseudouridine synthetase is as defined in claim 26.

30. A delivery composition comprising a delivery vector and one or more selected from the group consisting of: the tRNA mutant of any one of claims 1-18, the nucleic acid molecule of claim 19, the vector of claim 20, the composition of any one of claims 22-26, or the vector system of any one of claims 27-29; Preferably, the delivery composition comprises the composition of any one of claims 22-26; Preferably, the delivery composition comprises the carrier system of any one of claims 27-29.

31. A kit comprising the tRNA mutant of any one of claims 1 to 18, the nucleic acid molecule of claim 19, the vector of claim 20, the host cell of claim 21, the composition of any one of claims 22 to 26, the vector system of any one of claims 27 to 29, or the delivery composition of claim 30; Preferably, the delivery composition comprises the composition of any one of claims 22-26; Preferably, the delivery composition comprises the carrier system of any one of claims 27-29; Preferably, the kit comprises the delivery composition of claim 30; Preferably, the kit further comprises a first amino acid as defined in claim 22 or 23.

32. A translation system comprising: (a) a first component selected from: (i) a first O-tRNA selected from the tRNA mutant of any one of claims 1 to 18; (ii) a nucleotide sequence encoding the first O-tRNA; (iii) any combination of (i) and (ii); (b) a second component selected from the group consisting of: (i) a first O-RS, wherein the first O-RS is as defined in claim 22 or 23; (ii) a nucleotide sequence encoding the first O-RS; (iii) any combination of (i) and (ii); as well as, (c) a component III comprising a first amino acid as defined in claim 22 or 23; Preferably, the translation system comprises a host cell comprising the component I and the component II; Preferably, the translation system further comprises a nucleic acid molecule (eg, a DNA molecule or an RNA molecule) encoding a target protein, wherein the nucleic acid molecule comprises a selector codon (eg, an amber codon, an ochre codon, or an opal codon) that can be recognized by the first O-tRNA.

33. The translation system of claim 32, further comprising: (d) component IV selected from: (i) an engineered guide RNA as defined in claim 24 or 25; (ii) a nucleotide sequence encoding the engineered guide RNA; (iii) any combination of (i) and (ii); in, The guide sequence of the engineered guide RNA molecule is capable of hybridizing with the RNA molecule encoding the target protein.

34. The translation system of claim 33, further comprising: (e) Component V, which is selected from: (i) pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof); (ii) a nucleotide sequence encoding the pseudouridine synthase; (iii) any combination of (i) and (ii); in, The pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof) can be guided or recruited by the engineered guide RNA and modifies a uridine residue (e.g., a uridine residue in the selector codon, such as an amber codon, an ochre codon, or an opal codon) in an RNA molecule encoding the target protein that is hybridized with the engineered guide RNA into a pseudouridine residue; Preferably, the pseudouridine synthase is endogenous or exogenous to the host cell.

35. A method for producing a protein containing a target amino acid at a target position in a translation system, comprising: (1) Providing: a translation system according to any one of claims 32 to 34; (2) incorporating the target amino acid into the target position of the protein in response to the selector codon during translation of the protein, wherein the target position in the protein corresponds to the position of the selector codon in the nucleic acid molecule encoding the target protein, thereby producing a protein containing the target amino acid at the target position; Wherein, the target amino acid is the first amino acid recognized by the first O-tRNA.

36. A method for producing a protein containing a target amino acid at a target position in a host cell, comprising: (1) Providing a host cell, which comprises: (a) a first component selected from: (i) a first O-tRNA selected from the tRNA mutant of any one of claims 1 to 18; (ii) a nucleotide sequence encoding the first O-tRNA; (iii) any combination of (i) and (ii); (b) a second component selected from: (i) a first O-RS, wherein the first O-RS is as defined in claim 22 or 23; (ii) a nucleotide sequence encoding the first O-RS; (iii) any combination of (i) and (ii); and (c) a third component comprising a nucleic acid molecule (e.g., a DNA molecule or an RNA molecule) encoding a target protein, wherein the nucleic acid molecule comprises a selector codon (e.g., an amber codon, an ochre codon, or an opal codon) that is recognized by the first O-tRNA; (2) culturing the host cell in the presence of a target amino acid; wherein the target amino acid is the first amino acid recognized by the first O-tRNA; as well as, (3) incorporating the target amino acid into the target position of the protein during translation of the protein, wherein the target position in the protein corresponds to the position of the selector codon in the nucleic acid molecule, thereby producing a protein containing the target amino acid at the target position.

37. The method of claim 36, wherein The host cell further comprises: (d) a fourth component selected from: (i) an engineered guide RNA as defined in claim 24 or 25; (ii) a nucleotide sequence encoding the engineered guide RNA; (iii) any combination of (i) and (ii); The guide sequence of the engineered guide RNA molecule is capable of hybridizing with the RNA molecule encoding the target protein.

38. The method of claim 37, wherein The host cell further comprises: (e) a fifth component selected from the group consisting of: (i) a pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof); (ii) a nucleotide sequence encoding the pseudouridine synthase; (iii) any combination of (i) and (ii); wherein the pseudouridine synthase (e.g., DKC1 protein or a functional fragment thereof) can be guided or recruited by the engineered guide RNA and modifies the uridine residues (e.g., uridine residues in the selector codons, such as amber codons, ochre codons, or opal codons) in the RNA molecule encoding the target protein that hybridizes with the engineered guide RNA into pseudouridine residues; Preferably, the pseudouridine synthase is endogenous or exogenous to the host cell.

39. The method of any one of claims 36 to 38, wherein The first component, the second component, the third component, the fourth component, and the fifth component are each independently derived from endogenous expression or exogenous delivery of the host cell.

40. Use of the tRNA mutant of any one of claims 1 to 18, the nucleic acid molecule of claim 19, the vector of claim 20, the host cell of claim 21, the composition of any one of claims 22 to 26, the vector system of any one of claims 27 to 29, or the delivery composition of claim 30, the kit of claim 31 or the translation system of any one of claims 32 to 34 for producing a protein containing a target amino acid at a target position.

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