A site-specific modified engineered tRNA and its application

Abstract

A site-specific modified engineered tRNA and its application, wherein the modification is to replace one or more natural unmodified nucleotides selected from the 4th, 9th, 13th, 14th, 17th, 18th, 27th, 28th, 32nd, 34th, 35th, 36th, 37th, 38th, 39th, 49th, 50th, 54th, 55th, 58th, 72nd positions on the tRNA molecule with one or more modified nucleotides. Compared with the unmodified tRNA, the site-specific modified tRNA of the present invention has higher structural stability and / or stronger resistance to nuclease degradation, and the aminoacylation efficiency is not significantly reduced and the immunogenicity is not significantly increased. The modified tRNA can be used to eliminate the premature termination of translation caused by nonsense mutations, and has great application potential in the treatment of genetic diseases and tumors related to nonsense mutations. The modification of engineered tRNA is also of reference significance for tRNA drug development, and can guide the modification design of natural tRNA.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a site-specifically modified engineered tRNA and an application thereof, and in particular to a method for improving structural stability and decoding ability by modifying the engineered tRNA. Background Art

[0002] For a long time, scientists have developed drugs based on the flow of genetic information in the central dogma, and drugs targeting DNA, RNA and protein have emerged in an endless stream.

[0003] tRNAs were once considered to be highly abundant, ubiquitous, passive mRNA decoders and protein translation elements. The anticodon on the tRNA binds to the codon on the mRNA to carry the specified amino acid to complete protein translation. tRNAs that carry the same amino acid but different anticodons are called "isoacceptors"; tRNAs that carry the same amino acid and have the same anticodon but different sequences are "isodecoders". The latest scientific research progress unexpectedly discovered that tRNAisoacceptors and isodecoders have specific, non-passive translation-related active regulatory functions that can significantly affect biological processes and disease progression. As a result, scientists have gradually shifted their focus from mRNA to the new generation of RNA technology, exploring the potential of the new generation of RNA technology, and tRNA, which had not been paid attention to by the industry before, has stood out from it.

[0004] The full name of tRNA is transfer RNA. It is part of the cell's protein synthesis mechanism. tRNA can "read" the codons composed of three nucleotides on mRNA, and then transport the corresponding amino acids to the ribosomes and add them to the continuously generated polypeptide chains. This process will continue until the ribosome encounters a stop codon, at which time the ribosome will fall off the mRNA and complete the synthesis of the protein. However, in some cases, gene mutations will convert the codons that originally encode amino acids into stop codons, which leads to the premature termination of protein synthesis and the loss of function of the generated protein. It is estimated that about 11% of genetic diseases are caused by gene mutations that cause the stop codon to be premature and protein translation to terminate prematurely (the so-called nonsense mutations).

[0005] 61 codons in the human genome can be recognized by tRNA, encoding 20 amino acids. Three stop codons (UAG, UAA, UGA) do not have corresponding tRNA recognition, do not encode amino acids, and thus terminate translation. However, studies have found that there are tRNAs that can recognize stop codons, so that the stop codons encode amino acids and protein translation proceeds normally. This tRNA that can recognize stop codons is nonsense mutation engineered tRNA. Engineered tRNAs exist widely, and have been found in both plant cells and animal cells. Engineered tRNAs can overcome premature termination codons (PTC). After binding to PTC, they will not cause protein synthesis to stop, but will add the correct amino acid at the position of PTC, which allows protein synthesis to be completed smoothly. Therefore, for genetic diseases caused by PTC, in theory, only one engineered tRNA is needed to eliminate the premature interruption of translation caused by this nonsense mutation, thereby curing thousands of different genetic diseases.

[0006] In 1982, Yuet Wai Kan of the University of California, San Francisco, proposed a method to treat β-thalassemia by correcting the activity of pathogenic proteins through engineered tRNA. In the early 21st century, researchers at the University of Colorado, Boulder, verified the feasibility of this therapy in transgenic mice. Subsequently, researchers at the Ott Institute of Obstetrics and Gynecology in St. Petersburg, Russia, used engineered tRNA to correct the disease phenotype of the Duchenne muscular dystrophy mouse model. In 2014, researchers in Portugal showed that engineered tRNA has therapeutic prospects and preventive potential for hereditary tumor syndromes caused by nonsense mutations.

[0007] Currently, tRNA therapy is still in the exploratory stage, but the signs of industrialization have gradually emerged. ShapeTherapeutics, Tevard Biosciences, ReCode Therapeutics, Alltrna, hCBioscience, Qixia Decoding Bio and other companies are conducting research on the development of therapeutic tRNA, targeting diseases such as Rett syndrome, genetic epilepsy, Dravet syndrome, Duchenne muscular dystrophy, etc.

[0008] The number of tRNA nucleotides is between siRNA and mRNA, so its molecular weight is generally larger than ASO, siRNA, Aptamer, gRNA, but smaller than mRNA. Since the complex stem-loop structure of tRNA is more similar to aptamers, the diameters of the two are also similar. This suggests that tRNA can be used to draw on the drug-making strategies of other nucleic acid drugs ( Figure 1There are many studies on the modification and delivery of siRNA and mRNA1.

[0009] Although existing studies have shown that tRNA has good prospects for industrial application, in practical applications, there are still problems such as low decoding efficiency of tRNA for premature termination codons (PTC), poor stability, and safety that needs to be verified. Therefore, it is urgent to improve the structural stability and resistance to nuclease degradation of tRNA, reduce immunogenicity, and maintain aminoacylation efficiency. However, due to the low content of natural endogenous engineered tRNA in the body and the unclear factors affecting the decoding efficiency of PTC, the structural modification of tRNA is quite difficult. Summary of the invention

[0010] In view of the technical problems existing in the above-mentioned prior art, such as low decoding efficiency, poor stability and unverified safety of tRNA for premature termination codons (PTC), the purpose of the present invention is to provide a site-specific modified engineered tRNA, wherein the modification is to replace one or more natural unmodified nucleotides selected from positions 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58 and 72 on the tRNA molecule with one or more modified nucleotides; the modified nucleotides are selected from non-natural bases and naturally occurring modified bases, and the site-specific modified tRNA can carry natural amino acids and recognize premature termination codons (PTC). Compared with unmodified tRNA, the site-specific modified tRNA of the present invention has higher structural stability and / or stronger resistance to nuclease degradation, and the aminoacylation efficiency is not significantly reduced and the immunogenicity is not significantly increased. The modified tRNA can be used to eliminate premature translation termination caused by nonsense mutations, and thus has great application potential in treating genetic diseases and tumors related to nonsense mutations. The modification of tRNA is also of reference significance for tRNA drug development and can guide the modification design of natural tRNA.

[0011] Brief description of the invention

[0012] In one aspect, the present invention provides an engineered tRNA with site-directed modification, wherein the modification comprises replacing at least the natural nucleotide at position 34 of the tRNA molecule with a modified nucleotide;

[0013] The modified nucleotide is selected from at least one of a nucleotide having a non-natural base and a nucleotide having a natural modified base; the non-natural base includes at least one of X, Y, and Z; the natural modified base includes at least one of Um, Ψ, m1Ψ, m5Um, m1G, Gm, m1A, Cm, m5C, and m3C;

[0014] The site-specific modified tRNA can carry natural amino acids and recognize premature termination codons (PTCs).

[0015] Furthermore, the site-specific modified engineered tRNA of the present invention, the modified engineered tRNA also includes modification of one or more sites selected from the following: tRNA molecule (positions 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58, 72); the modification of one or more sites is to replace the natural nucleotides of one or more sites with modified nucleotides.

[0016] Furthermore, in the site-specifically modified engineered tRNA of the present invention, the modified nucleotide at position 34 of the tRNA is Cm (2'-O-Methylcytidine) or Um (2'-O-Methyluridine).

[0017] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying the following amino acids: at least one of glutamine (Gln), tryptophan (Trp), tyrosine (Tyr), and leucine (Leu).

[0018] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying tryptophan (Trp), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 34 with Cm, and replacing the nucleotide at position 35 with Ψ.

[0019] Furthermore, in the site-directed modified engineered tRNA of the present invention, the tRNA is selected from tRNA carrying tyrosine (Tyr), and the modification of the tRNA molecule is to replace one site or multiple sites simultaneously: the nucleotide at position 34 is replaced by a 2'-O-methylated modified nucleotide such as Cm and Um, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 37 is replaced by m1G, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotide at position 58 is replaced by m1A, the nucleotides at positions 18 and 34 are replaced by Gm and Cm, and the nucleotides at positions 18 and 55 are replaced by Gm and Ψ.

[0020] Further, in the site-directed modified engineered tRNA of the present invention, the tRNA is selected from tRNA carrying leucine (Leu), and the modification of the tRNA molecule is to replace one site or multiple sites simultaneously: the nucleotide at position 34 is replaced by a 2'-O-methylated modified nucleotide such as Cm and Um, the nucleotide at position 9 is replaced by m1G, the nucleotide at position 17 is replaced by Gm, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 37 is replaced by m1G, the nucleotide at position 38 is replaced by Ψ, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotides at positions 17 and 34 are replaced by Gm and Cm, and the nucleotides at positions 17 and 55 are replaced by Gm and Ψ, nucleotides at positions 32 and 38 were replaced by Cm and Ψ, and nucleotides at positions 34 and 55 were replaced by Cm and Ψ.

[0021] Further, the site-directed modified engineered tRNA of the present invention, the tRNA is a tRNA carrying glutamine (Gln), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 9 with m1G, replacing the nucleotide at position 49 with m5C, replacing the nucleotide at position 39 with Um or Ψ, replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with a 2'-O-methylated modified nucleotide such as Cm and Um, replacing the nucleotides at positions 18 and 34 with Gm and Cm respectively, replacing the nucleotide at position 4 with Um, and replacing the nucleotide at position 13 with The nucleotide at position 14 is replaced by m1A, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 27 is replaced by m5C, the nucleotide at position 28 is replaced by Ψ, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 36 is replaced by Z, the nucleotide at position 37 is replaced by a non-natural base X or Y, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotide at position 58 is replaced by m1A, the nucleotide at position 72 is replaced by m5C, and the nucleotides at positions 18 and 55 are replaced by Gm and Ψ.

[0022] Furthermore, the site-specific modified engineered tRNA of the present invention comprises modifications at the following two sites: the nucleotide at position 18 is replaced by Gm, and the nucleotide at position 34 is replaced by a 2'-O-methylated modified nucleotide, such as Cm / Um.

[0023] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying serine (Ser), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Cm or Um, and replacing the nucleotide at position 35 with Ψ.

[0024] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying lysine (Lys), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Cm, and replacing the nucleotide at position 35 with Ψ.

[0025] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying arginine (Arg), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Um or Ψ, and replacing the nucleotide at position 55 with Ψ.

[0026] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying glycine (Gly), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Um or Ψ, and replacing the nucleotide at position 55 with Ψ.

[0027] Furthermore, the site-specific modified engineered tRNA of the present invention is a tRNA carrying cysteine ​​(Cys), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Um or Ψ, and replacing the nucleotide at position 55 with Ψ.

[0028] In another aspect, the present invention provides a method for improving the translation activity of tRNA and / or enhancing the decoding efficiency of tRNA, wherein at least the 34th nucleotide of tRNA is replaced with a 2'-O-methylated nucleotide; the tRNA is selected from tRNA Gln(CTA) tRNA Trp(CTA) tRNA Tyr(CTA) tRNA Leu(CTA) tRNA Ser(CTA) tRNA Glu(CTA) tRNA Lys(CTA) tRNA Arg(TCA) tRNA Gly(TCA) tRNA Cys(TCA) tRNA Leu(TCA) tRNA Ser(TCA)tRNA Leu(TTA) tRNA Gln(TTA) tRNA Tyr(TTA) tRNA Ser(TTA) tRNA Gln-CTG The present invention also provides the use of the site-directed modified engineered tRNA in correcting nonsense mutations caused by premature termination codons (PTCs). DETAILED DESCRIPTION OF THE INVENTION

[0030] In one aspect, the present invention provides an engineered tRNA with site-directed modification, wherein the modification comprises replacing at least the natural nucleotide at position 34 of the tRNA molecule with a modified nucleotide;

[0031] The modified nucleotide is selected from at least one of a nucleotide having a non-natural base and a nucleotide having a natural modified base; the non-natural base includes at least one of X, Y, and Z; the natural modified base includes at least one of Um, Ψ, m1Ψ, m5Um, m1G, Gm, m1A, Cm, m5C, and m3C;

[0032] The site-specific modified tRNA can carry natural amino acids and recognize premature termination codons (PTCs).

[0033] The nucleotide numbering positions on the tRNA molecule of the present invention are well known in the art, and specific reference is made to Figure 4a Although the sequence lengths of tRNAs in the prior art are not exactly the same, they all have Figure 4a The secondary structure shown in FIG. Figure 4a The nucleotide number position in determines the nucleotide site of the tRNA.

[0034] On the other hand, the site-specific modified engineered tRNA of the present invention further comprises modification of one or more sites selected from the following: tRNA molecule 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58, 72; the modification of the one or more sites is to replace the natural nucleotides of the one or more sites with modified nucleotides;

[0035] The modified nucleotide is selected from at least one of a nucleotide having a non-natural base and a nucleotide having a natural modified base; the non-natural base includes at least one of X, Y, and Z; the natural modified base includes at least one of Um, Ψ, m1Ψ, m5Um, m1G, Gm, m1A, Cm, m5C, and m3C.

[0036] Preferably, in the site-specific modified engineered tRNA of the present invention, the nucleotide modified at position 34 of the tRNA is a 2'-O-methylated nucleotide, and the 2'-O-methylated nucleotide is selected from at least one of Cm, Um, and Gm.

[0037] Preferably, the site-specific modified engineered tRNA of the present invention is a tRNA carrying the following amino acids: at least one of glutamine (Gln), tryptophan (Trp), tyrosine (Tyr), leucine (Leu), serine (Ser), lysine (Lys), glutamate (Glu), arginine (Arg), cysteine ​​(Cys), and glycine (Gly).

[0038] In a specific embodiment, the tRNA is a tRNA carrying tryptophan (Trp), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 34 with a 2'-O-methylated modified nucleotide such as Cm and Um, replacing the nucleotide at position 35 with Ψ, replacing the nucleotides at positions 34 and 35 with Cm and Ψ, replacing the nucleotide at position 9 with m1G, replacing the nucleotide at position 17 with Gm, replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 32 with m3C, replacing the nucleotide at position 36 with Z, replacing the nucleotide at position 37 with m1G or X and Y, replacing the nucleotide at position 39 with Ψ or Um, replacing the nucleotide at position 54 with Um or m5Um, replacing the nucleotide at position 55 with Ψ or Um, replacing the nucleotide at position 34 with Cm, replacing the nucleotide at position 34 with Cm, replacing the nucleotides at positions 17 and 34 with Gm and Cm, and replacing the nucleotides at positions 18 and 34 with Gm. and Cm, and nucleotides at positions 34 and 55 were replaced by Cm and Ψ.

[0039] In a specific embodiment, the tRNA is selected from tRNA carrying tyrosine (Tyr) and the modification of the tRNA molecule is to replace one or more sites simultaneously: the nucleotide at position 34 is replaced by a 2'-O-methylated modified nucleotide such as Cm and Um, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 37 is replaced by m1G, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotide at position 58 is replaced by m1A, the nucleotides at positions 18 and 34 are replaced by Gm and Cm, and the nucleotides at positions 18 and 55 are replaced by Gm and Ψ.

[0040] In a specific embodiment, the tRNA is selected from tRNA carrying leucine (Leu), and the modification of the tRNA molecule is to replace one or more sites simultaneously: the nucleotide at position 34 is replaced by a 2'-O-methylated modified nucleotide such as Cm and Um, the nucleotide at position 9 is replaced by m1G, the nucleotide at position 17 is replaced by Gm, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 37 is replaced by m1G, the nucleotide at position 38 is replaced by Ψ, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotides at positions 17 and 34 are replaced by Gm and Cm, the nucleotides at positions 17 and 55 are replaced by Gm and Ψ, the nucleotides at positions 32 and 38 are replaced by Cm and Ψ, and the nucleotides at positions 34 and 55 are replaced by Cm and Ψ.

[0041] In a specific embodiment, the tRNA is a tRNA carrying glutamine (Gln), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 9 with m1G, replacing the nucleotide at position 49 with m5C, replacing the nucleotide at position 39 with Um or Ψ, replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with a 2'-O-methylated modified nucleotide such as Cm and Um, replacing the nucleotides at positions 18 and 34 with Gm and Cm, respectively, replacing the nucleotide at position 4 with Um, replacing the nucleotide at position 13 with Ψ, and replacing the nucleotide at position 14 with Ψ. The nucleotide at position 4 is replaced by m1A, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 27 is replaced by m5C, the nucleotide at position 28 is replaced by Ψ, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 36 is replaced by Z, the nucleotide at position 37 is replaced by a non-natural base X or Y, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotide at position 58 is replaced by m1A, the nucleotide at position 72 is replaced by m5C, and the nucleotides at positions 18 and 55 are replaced by Gm and Ψ.

[0042] In a specific embodiment, the tRNA is a tRNA carrying serine (Ser), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Cm or Um, and replacing the nucleotide at position 35 with Ψ.

[0043] In a specific embodiment, the tRNA is a tRNA carrying lysine (Lys), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Cm, and replacing the nucleotide at position 35 with Ψ.

[0044] In a specific embodiment, the tRNA is a tRNA carrying arginine (Arg), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Um or Ψ, and replacing the nucleotide at position 55 with Ψ.

[0045] In a specific embodiment, the tRNA is a tRNA carrying glycine (Gly), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Um or Ψ, and replacing the nucleotide at position 55 with Ψ.

[0046] In a specific embodiment, the tRNA is a tRNA carrying cysteine ​​(Cys), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Um or Ψ, and replacing the nucleotide at position 55 with Ψ.

[0047] On the other hand, the present invention also provides use of the site-directed modified engineered tRNA in correcting nonsense mutations caused by premature termination codons (PTCs).

[0048] On the other hand, the present invention also provides a method for improving the translation activity of tRNA and / or enhancing the decoding efficiency of tRNA, wherein at least the 34th nucleotide of tRNA is replaced with a 2'-O-methylated nucleotide; the tRNA is selected from tRNA Gln(CTA) tRNA Trp(CTA) tRNA Tyr(CTA) tRNA Leu(CTA) tRNA Ser(CTA) tRNA Glu(CTA) tRNA Lys(CTA) tRNA Arg(TCA) tRNA Gly(TCA) tRNA Cys(TCA) tRNA Leu(TCA) tRNA Ser(TCA) tRNA Leu(TTA) tRNA Gln(TTA) tRNA Tyr(TTA) tRNA Ser(TTA) tRNA Gln-CTG The heterogeneous decoding family (isodecoder).

[0049] On the other hand, the present invention also provides a method for improving tRNA translation activity and / or enhancing tRNA decoding efficiency, comprising:

[0050] Modifying one or more sites on the tRNA molecule selected from the group consisting of: positions 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58, 72; wherein the modification of the one or more sites is to replace the natural nucleotides at the one or more sites with modified nucleotides;

[0051] The modified nucleotide is selected from at least one of a nucleotide having a non-natural base and a nucleotide having a natural modified base; the non-natural base includes at least one of X, Y, and Z; the natural modified base includes at least one of Um, Ψ, m1Ψ, m5Um, m1G, Gm, m1A, Cm, m5C, and m3C.

[0052] Advantages and beneficial effects of the present invention:

[0053] The present invention first draws on the modification paradigm of natural tRNA, selects modification sites in combination with the results of 3D simulation of tRNA molecular structure, evaluates the effect of each modification site on tRNA performance, and obtains modification sites and modification patterns that are favorable to tRNA performance. By chemically synthesizing tRNAs carrying four amino acids, the effects of the favorable modification sites and modification patterns obtained in the initial screening on the translation activity of tRNA are verified. The modification sites and modification patterns that are most favorable to translation activity are selected to further verify their decoding efficiency for PTC.

[0054] First, the present invention confirms that naturally occurring tRNA modification sites and modification patterns are not always beneficial to structural stability, tRNA translation activity, and PTC decoding read-through efficiency. Considering structural stability alone may also affect the binding of tRNA to aminoacyl-tRNA synthetase. However, in addition to translation activity, factors such as nuclease resistance and immunogenicity should also be considered for comprehensive consideration.

[0055] Second, the present invention found that although the same modification at the same site in tRNA molecules carrying different amino acids produced different effects, the Cm modification at position 34 showed better structural stability, higher translation activity, and stronger read-through efficiency in both tRNAs carrying different amino acids and tRNA heterologous decoding family molecules carrying the same amino acids.

[0056] Third, the present invention uses the GFP mRNA of UAG at position 39 studied in the previous study as a reporter system to study the function of modified tRNA, which more truly reflects the biological function of tRNA in cells. The research method is simple and fast, and the results obtained are more real and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0058] Figure 1 : Comparison of diameters and molecular weights of commonly used nucleic acid therapeutics.

[0059] Figure 2 :Modification site of tRNA and modified nucleotide.

[0060] Figure 3 : In tRNA Gln or tRNA Trp Modified nucleotides are introduced completely or randomly, wherein:

[0061] (a) Ψ, m1Ψ, and Z-tRNA with different ratios of modified nucleotides Gln Fluorescence results of HEK293T cells co-transfected with GFP-UAG mRNA;

[0062] (b) Chemical structural formulas of Ψ, m1Ψ, Z, dX, dY, X, Y;

[0063] (c)37X, Y-tRNA Gln , 37X, Y-tRNA Trp Fluorescence results of HEK293T cells co-transfected with GFP-UAG mRNA.

[0064] Figures 4a to 4e :Chemical synthesis of site-specific modified tRNA Gln , tRNA Tyr , tRNA Leu and tRNA Trp ,in:

[0065] Figure 4a :The factors to be considered for introducing tRNA modifications and the sites for introducing modifications are summarized. The red fonts in the table represent endogenous modifications in nature involved in this experiment; the blue fonts represent artificial modifications or non-natural base modifications in nucleic acid drugs involved in this experiment;

[0066] Figure 4b : tRNA Gln Fluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UAG mRNA;

[0067] Figure 4c : tRNA TrpFluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UAG mRNA;

[0068] Figure 4d : tRNA Tyr Fluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UAG mRNA;

[0069] Figure 4e : tRNA Leu Fluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UAG mRNA.

[0070] Figure 5 : Chemical synthesis of site-specific modified tRNAs that read through three nonsense mutation codons, wherein:

[0071] (a) Modified tRNA Ser(CTA) , tRNA Lys(CTA) , tRNA Glu(CTA) Fluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UAG mRNA;

[0072] (b) Modified tRNA Gln(TTA) , tRNA Leu(TTA) , tRNA Tyr(TTA) , tRNA Ser(TTA) Fluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UAAmRNA;

[0073] (c) Modified tRNA Arg(TCA) , tRNA Gly(TCA) , tRNA Cys(TCA) , tRNA Leu(TCA) , tRNA Ser(TCA) Fluorescence images at 24 h intervals after HEK293T cells were co-transfected with GFP-UGA mRNA;

[0074] (d) tRNA Gln(CTA) Fluorescence images and flow cytometry analysis of isodecoders 34-position 2'-O-methylated and co-transfected with GFP-UAG mRNA into HEK293T cells at 24h intervals;

[0075] (e) tRNA Gln(CTA) , tRNA Tyr(CTA) , tRNA Trp(CTA) , tRNA Leu(CTA) , tRNA Ser(CTA) , tRNA Lys (CTA) , tRNA Glu(CTA), tRNA Gln(TTA) , tRNA Leu(TTA) , tRNA Tyr(TTA) , tRNA Ser(TTA) , tRNA Arg(TCA) ,tRNA Gly(TCA) , tRNA Cys(TCA) , tRNA Leu(TCA) , tRNA Ser(TCA) After 2'-O-methylation modification at position 34, the mRNA was co-transfected with GFP-UAG / UAA / UGA mRNA into HEK293T cells and analyzed by flow cytometry;

[0076] Figure 6 :Characterization and application of chemically synthesized 34Cm-modified, Cy5-modified, and GalNac-modified tRNA.

[0077] Figure 6 Middle: (a) tRNA is transported via LNP Tyr(CTA) , tRNA Tyr(CTA) -34Cm, tRNA Ser(CTA) , tRNA Ser(CTA) -34Cm, tRNA Gln(CTA) , tRNA Gln(CTA) -34Cm, tRNA Leu(CTA) , tRNA Leu(CTA) -34Cm, tRNA Leu(CTA) -17Gm+34Cm transfected MPS IH nonsense mutant mice (B6.129S- Idua tm1.1Kmke / J) fibroblasts, wherein the IDUA gene of the fibroblasts comprises a TAG mutation at position 392;

[0078] (b) In tRNA Gln(CTA) -34Cm 5', 3' end and D-loop were labeled with Cy5, and HEK293T cells were co-transfected with GFP-UAG mRNA for 24 hours. Note: 5'-Cy5-Gln(CTA)-34Cm, 3'-Cy5-Gln(CTA)-34Cm, D-Cy5-Gln(CTA)-34Cm;

[0079] (c) 3'-Cy5-tRNA modified with GalNac at the 5' and 3' ends Gln(CTA) -34Cm or D-Cy5-tRNA Gln(CTA) Fluorescence image of HEK293T cells co-transfected with -34Cm and GFP-UAG mRNA for 24 hours. Note: 5'-GalNac-D-Cy5-tRNA Gln (CTA)-34Cm: D-Cy5-Gln(CTA)-34Cm with GalNac modification at the 5' end, 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: D-Cy5-Gln(CTA)-34Cm with GalNac at the 3' end, 5'-GalNac-3'-Cy5-tRNA Gln(CTA) -34Cm: 3'-Cy5-Gln (CTA)-34Cm with GalNac modification at the 5' end;

[0080] (d) Using 0.9% saline as a control, polyplus (Cat. No.: 101000040) and LNP (LipidNanoparticle, SM102) were used to deliver D-Cy5-tRNA. Gln(CTA) -34Cm, tail vein injection (IV), intramuscular injection (IM), intraperitoneal injection (IP), compare the distribution of tRNA;

[0081] (e) Using 0.9% saline as control, D-Cy5-tRNA was delivered by LNP Gln(CTA) and D-Cy5-tRNA Gln (CTA) -34Cm, injected into mice via tail vein (IV). Comparison of distribution time before and after modification;

[0082] (f) Subcutaneous injection of 5'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm and 3'-GalNac-D-Cy5-tRNA Gln (CTA) -34Cm. DETAILED DESCRIPTION

[0083] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention. Rather than all embodiments, it should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present invention.

[0084] Unnatural base pairs are important elements for building synthetic life. Life on Earth evolved from a common ancestor and shares a set of genetic codes. This biological information is stored in DNA, which is composed of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC) and deoxythymidine (dT). This information is stored and retrieved by forming two base pairs (d)A-dT / U and (d)G-(d)C. Scientists have developed unnatural base pairs (UBPs) beyond the four bases, injecting new elements into the storage and transmission of genetic information. The process of DNA replication, RNA transcription, and translation has been opened up. This study used dNaM(dX)-dTPT3(dY) and NaM(X)-TPT3(Y) base pairs to introduce unnatural bases NaM(X) or TPT3(Y) into the 37th position of tRNA. 9 Due to the resistance of unnatural bases to endogenous nucleases, it is hoped that the stability of the engineered tRNA produced will be enhanced. 2'-aminoadenine (Z) is also an unnatural base that was found in Bacteriophage 2 Z has an additional amino group compared to natural adenine, which theoretically enhances the interaction between bases. Zhang M et al. reported that the Z-mRNA produced by incorporating it into mRNA has higher translation ability, lower cytotoxicity and lower immunogenicity compared to unmodified mRNA. 3 Pseudouridine (Ψ), Methylguanosine (Gm), 2'-O-Methylcytidine (Cm), 2'-O-Methyluridine (Um), 5-Methyluridine (m5U), 3-Methylcytidine (m3C), 1-Methyladenosine (m1A), 5-Methylcytidine (m5C), 1-Methylguanosine (m1G) are also potential alternative tRNA modifications. Ψ is a commonly used modification in mRNA vaccines that can reduce the immunogenicity of mRNA. 4 Similarly, introducing Ψ into tRNA is expected to reduce the immunogenicity of exogenously transferred tRNA. However, if all U replaces Ψ in mRNA, the higher-order structure of tRNA may be destroyed, resulting in it no longer functioning. Gm, Cm, and Um are common ribose 2'-O-methylation modifications, which are common in siRNA modifications and help resist nucleases and reduce immune stimulation to a certain extent. These modifications are also common modifications in endogenous tRNA and are related to immune recognition, decoding efficiency, etc. m5U, m3C, m1A, m5C, and m1G are common modifications of endogenous tRNA. 5 , m5C, m5U, m1A4,6 These are also modified nucleotides that have been screened for mRNA vaccines, indicating that these modifications are promising as potential candidates.

[0085] The modifications in the present invention mainly involve non-natural bases X, Y, Z, common nucleic acid drug modifications and endogenous RNA modifications Um, Ψ, m1Ψ, m5U, m1G, Gm, m1A, Cm, m5C, m3C. The modified nucleotides on tRNA involved in the present invention are as follows Figure 2 As shown in the figure, more than one modified nucleotide is designed at positions 34, 37, 39, 54, and 55, and their structural formulas are listed. The introduction of modified nucleotides follows the principles of enhancing tRNA decoding activity, enhancing stability, and reducing immune stimulation. They are selected from non-natural base pairs in synthetic biology, modified nucleotides of nucleic acid drugs, and modified nucleotides of natural tRNA itself to obtain potential modified nucleotides, thereby screening for ideal modification paradigms. In addition, considering the effectiveness and safety issues in practical applications, although some modifications will not enhance the translation activity of tRNA, they may reduce its immune activity and enhance nuclease resistance. Therefore, if the modified tRNA has translation activity, it should be considered as a beneficial modification.

[0086] Example 1: Introduction of unnatural bases into in vitro transcribed IVT-tRNA

[0087] Based on previous studies, GFP mRNA containing UAG was used as a reporter system. 7 When preparing tRNA, refer to the preparation of in vitro transcribed mRNA and select T7-tRNA Gln As a transcription template, replace all U or A in the in vitro transcription system with Ψ 4 , m 1 Ψ 8 and Z 3 ( Figure 3 (a)), it was found that the Ψ, m1Ψ, Z-tRNA obtained by in vitro transcription Gln The decoding activity is low. Gln For example, the incorporation ratio of Z was gradually changed from 0:1 to 1:1 and 1.5:0.5. It was found that when A:Z=1.5:0.5, GFP expression was restored and weak fluorescence could be detected.

[0088] In addition, we attempted to introduce unnatural bases into tRNA using unnatural base pairs ( Figure 3 (b)

[0089] We selected the dNaM(dX)-dTPT3(dY) and NaM(X)-TPT3(Y) base pairs 9Given the hydrophobicity of X and Y, they were chosen to be inserted into tRNA Gln and tRNA Trp Position 37 of the mRNA. This position is adjacent to the anticodon, and modification of this site helps ensure that the mRNA 10,11 Accurate decoding and maintaining the correct reading frame. Helps maintain the anticodon loop structure 12,13 , promoting the interaction between tRNA and mRNA and rRNA. The hydrophobic structure of the purine modification at position 37 theoretically helps to disrupt the interaction between 33U-37A and form the U turn of the anticodon loop. 12–14 37X-tRNA Gln The expression of GFP-UAG was restored, providing a starting construct for subsequent optimization.

[0090] Example 2: In chemically synthesized tRNA Gln , tRNA Tyr tRNA Leu tRNA Trp Endogenous modifications

[0091] Phosphoramidite monomers can be used to synthesize full-length tRNA in the solid phase and incorporate modified nucleotides at specific sites. This method provides the possibility of high-throughput screening of beneficial modification sites. The introduction of modifications is to reduce the immunogenicity of tRNA 16–18 , enhancing its nuclease resistance and thus prolonging its action time, serving as a recognition element for aminoacyl-tRNA synthetases 19,20 , stabilizes the tRNA structure 12 , promoting the binding of metal ions 11 , fine-tuning tRNA structure to promote interaction between mRNA and rRNA to enhance decoding efficiency 13 .

[0092] Here, based on our understanding of existing tRNA modifications, we adopted an engineering approach to first screen for single-site modifications and further evaluate dual-site modifications, providing methods and ideas for subsequent mechanism studies and drugability optimization. We combined the endogenous modification spectrum of tRNA 5 The functions of the various parts of tRNA structure, the relationship between tRNA modification and tRF 25 , siRNA modification substitutions, and possible strategies for the introduction of tRNA modifications were mapped ( Figure 4a ).

[0093] For tRNA GlnFor example, modifications of 9m1G, 18Gm, 34Cm, 39Ψ, 39Um, 49m5C, 54Um, 55Ψ, and double-site 2'-O-methylation substitutions at positions 18 and 34 can all improve translation activity. 18Gm is an important modification that distinguishes prokaryotic and eukaryotic tRNAs. Bacterial tRNAs can escape immunity after acquiring 18Gm modification. 17 , 34Cm may slightly reduce immune stimulation 28,29 , resisting nuclease cleavage. The modification of T / D-loop can theoretically help tRNA form a more stable tertiary structure. 12 .

[0094] For tRNA Trp For example, unmodified tRNA Trp The translation activity was low, but modifications of 17Gm, 18Gm, 34Cm, 35Ψ, 37m1G, 54m5Um, 55Ψ, 17Gm+34Cm, 18Gm+34Cm, and 34Cm+55Ψ increased its translation activity ( Figure 4c ), modification of both 34Cm and 35Ψ sites greatly enhanced the translation activity.

[0095] For tRNA Tyr For example, 34Cm, 35Ψ, and 58m1A increased its translation activity ( Figure 4d ).

[0096] For tRNA Leu For example, 34Cm, 35Ψ, 38Ψ, 55Ψ, and 17Gm+34Cm increased their translation activity ( Figure 4e ).

[0097] Example 3: 2'-O-methylation modification at position 34 affects the decoding efficiency of tRNA

[0098] In addition to the above four amino acids, the modification paradigm is similar for tRNA corresponding to other amino acids.

[0099] Figure 5 (a), (b), and (c) show tRNA Ser(CTA) , tRNA Lys(CTA) , tRNA Glu(CTA) , tRNA Gln(TTA) ,tRNA Leu(TTA) , tRNA Tyr(TTA) , tRNA Ser(TTA) , tRNA Arg(TCA) , tRNA Gly(TCA) , tRNA Cys(TCA) , tRNA Leu (TCA) , tRNASer(TCA) Changes in translation activity before and after modification. Arg(TCA) It is relatively sensitive to modification, and 55Ψ does not impair its translational activity ( Figure 5 (c) tRNA Ser(CTA) 18Gm did not affect its translation activity, but 34Cm and 35Ψ both effectively enhanced its translation activity ( Figure 5 (a)

[0100] Figure 6 tRNA Gln(CTA) , tRNA Tyr(CTA) , tRNA Trp(CTA) , tRNA Leu(CTA) , tRNA Ser(CTA) ,tRNA Lys(CTA) , tRNA Glu(CTA) , tRNA Gln(TTA) , tRNA Leu(TTA) , tRNA Tyr(TTA) , tRNA Ser(TTA) , tRNA Arg (TCA) , tRNA Gly(TCA) , tRNA Cys(TCA) , tRNA Leu(TCA) , tRNA Ser(TCA) Changes in translational activity after 2'-O-methylation at position 34. tRNA Ser(CTA) , tRNA Glu(CTA) , tRNA Leu(TCA) , tRNA Ser(TCA) , tRNA Leu(UUA) 2'-O-methylation modification at position 34 can significantly enhance its translation activity ( Figure 5 (e)

[0101] In addition, tRNA Gln-CTG The 2'-O-methylation modification of position 34 of the six tRNAs with translational activity can significantly improve their translation efficiency ( Figure 5 (d)).

[0102] Example 4: Cy5 labeling can trace tRNA distribution, and GalNac modification facilitates tRNA delivery

[0103] To further explore the translational activity and possible mechanism of 2'-O-methylation modification, B6.129S- Idua tm1.1Kmke / Fibroblasts from mice with J nonsense mutation disease were transfected with tRNA before and after 2'-O-methylation at position 34 Tyr(CTA) , tRNASer(CTA) , tRNA Gln(CTA) , tRNA Leu(CTA) , protein expression differences were analyzed after 24 h ( Figure 6 We found that compared with the protein expression in the control group, the IDUA protein recovery in the experimental group transfected with tRNA was increased, among which tRNA Tyr(CTA) The improvement after modification with 2'-oxymethyl at position 34 was more obvious.

[0104] In tRNA Gln(CTA) -34Cm's 3' end, 5' end and D-loop 16-position labeled with Cy5 dye, recorded as: 5'-Cy5-Gln(CTA)-34Cm, 3'-Cy5-Gln(CTA)-34Cm, D-Cy5-Gln(CTA)-34Cm. Verify that the three have translational activity ( Figure 6 (b)). In 3'-Cy5-tRNA Gln(CTA) -34Cm or D-Cy5-tRNA Gln(CTA) -34Cm 5' and 3' ends are modified with GalNac. Note: 5'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: D-Cy5-Gln (CTA)-34Cm with GalNac modification at the 5' end, 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: D-Cy5-Gln(CTA)-34Cm with GalNac at the 3' end, 5'-GalNac-3'-Cy5-tRNA Gln(CTA) -34Cm: 3'-Cy5-Gln(CTA)-34Cm with GalNac modification at the 5' end. The three were verified to have translation activity ( Figure 6 (c)).

[0105] Using 0.9% saline as a control, polyplus and LNP were used to deliver D-Cy5-Gln(CTA)-34Cm, and the distribution and retention of tRNA in mice can be reflected by detecting the Cy5 signal ( Figure 6 (d)). tRNA injected intravenously using LNP Gln(CTA) -34Cm compared to unmodified tRNA Gln(CTA) , and remained in the mouse body for a longer time ( Figure 6 This indicates that 2'-O-methylation at position 34 can enhance tRNA stability, prolong its action time and enhance its translation activity.

[0106] Subcutaneous injection of 5'-GalNac-D-Cy5-tRNA Gln(CTA)-34Cm and 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm, 12 h later, tRNA modified with 5' end GalNac was observed to accumulate in the abdominal cavity, and GalNac-modified tRNA can be directly delivered to treat nonsense mutation diseases.

[0107] By learning from tRNA modifications in nature, we can optimize the modification and delivery of tRNA drugs. In the future, we will further optimize the modification paradigm of tRNA, such as trying to introduce more non-endogenous modifications to enhance its anti-nuclease properties to prolong its action time in the body without affecting the translation activity of tRNA too much.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. The scope of patent protection of the present invention shall be subject to the claims described.

[0109] The sequence structure of the modified tRNA synthesized by the present invention is as follows:

[0111] The following prior art is fully incorporated into the specification by reference:

[0112] 1. Egli, M.&Manoharan, M. Chemistry, structure and function ofapproved oligonucleotide therapeutics. Nucleic Acids Res. 51, 2529–2573(2023).

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[0114] 3. Zhang, M., Singh, N., Ehmann, M. E., Zheng, L.&Zhao, H.Incorporation of noncanonical base Z yields modified mRNA with minimalimmunogenicity and improved translational capacity in mammalian cells.iScience 26, 107739 (2023).

[0115] 4. Karikó, K. et al. Incorporation of Pseudouridine Into mRNA YieldsSuperior Nonimmunogenic Vector With Increased Translational Capacity andBiological Stability. Mol. Ther. 16, 1833–1840 (2008).

[0116] 5. Suzuki, T. The expanding world of tRNA modifications and theirdisease relevance. Nat. Rev. Mol. Cell Biol. 22, 375–392 (2021).

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[0118] 7. Shi, N. et al. Restoration of dystrophin expression in mice bysuppressing a nonsense mutation through the incorporation of unnatural aminoacids. Nat. Biomed. Eng. 6, 195–206 (2022).

[0119] 8. Andries, O. et al. N1-methylpseudouridine-incorporated mRNAoutperforms pseudouridine-incorporated mRNA by providing enhanced proteinexpression and reduced immunogenicity in mammalian cell lines and mice. J.Controlled Release 217, 337–344 (2015).

[0120] 9. Feldman, A. W. et al. Optimization of Replication, Transcription,and Translation in a Semi-Synthetic Organism. J. Am. Chem. Soc. 141, 10644–10653 (2019).

[0121] 10. Björk, G. R., Wikström, P. M.&Byström, A. S. Prevention oftranslational frameshifting by the modified nucleoside 1-methylguanosine.Science 244, 986–989 (1989).

[0122] 11. Han, L.&Phizicky, E. M. A rationale for tRNA modificationcircuits in the anticodon loop. RNA 24, 1277–1284 (2018).

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[0124] 13. Agris, P. F. Bringing order to translation: the contributions oftransfer RNA anticodon‐domain modifications. EMBO Rep. 9, 629–635 (2008).

[0125] 14. Agris, P. Wobble position modified nucleosides evolved to selecttransfer RNA codon recognition: A modified-wobble hypothesis. Biochimie 73,1345–1349 (1991).

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[0128] 17. Freund, I. et al. 2′- O -methylation within prokaryotic andeukaryotic tRNA inhibits innate immune activation by endosomal Toll-likereceptors but does not affect recognition of whole organisms. RNA 25, 869–880(2019).

[0129] 18. Keller, P. et al. Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-likereceptor 7. Nucleic Acids Res. 46, 9764–9775 (2018).

[0130] 19. Muramatsu, T. et al. Codon and amino-acid specificities of atransfer RNA are both converted by a single post-transcriptionalmodification. Nature 336, 179–181 (1988).

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Claims

1. A site-specific modified engineered tRNA, characterized in that: The modification includes at least 2'-O-methylation modification of the natural nucleotide at position 34 of the natural tRNA molecule; the natural tRNA molecule is tRNA Ser(CTA) , tRNA Glu(CTA) , tRNA Leu(TCA) , tRNA Ser(TCA) , tRNA Leu(TTA) , tRNA Gln(CTA) ; When the natural tRNA molecule is tRNA Ser(CTA) When the tRNA Ser(CTA) The nucleotide sequence is SEQ ID NO:96, and the modified nucleotide at position 34 is Cm; When the natural tRNA molecule is tRNA Glu(CTA) When the tRNA Glu(CTA) The nucleotide sequence is SEQ ID NO: 104, and the modified nucleotide at position 34 is Cm; When the natural tRNA molecule is tRNA Leu(TCA) When the tRNA Leu(TCA) The nucleotide sequence is SEQ ID NO: 136, and the modified nucleotide at position 34 is Um; When the natural tRNA molecule is tRNA Ser(TCA) When the tRNA Ser(TCA) The nucleotide sequence is SEQ ID NO: 138, and the modified nucleotide at position 34 is Um; When the natural tRNA molecule is tRNA Leu(TTA) When the tRNA Leu(TTA) The nucleotide sequence is SEQ ID NO: 138, and the modified nucleotide at position 34 is Um; When the natural tRNA molecule is tRNA Gln(CTA) When the tRNA Gln(CTA) The nucleotide sequence is selected from SEQ ID NO: 1, 25, 27, 29, 31, 33, 96, and the modified nucleotide at position 34 is Cm.

2. The engineered tRNA for site-directed modification according to claim 1, characterized in that The modified engineered tRNA also includes modification of one or more sites selected from the following: tRNA molecule sites 4, 9, 13, 14, 17, 18, 27, 28, 32, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58, 72; the modification of the one or more sites is to replace the natural nucleotides at the one or more sites with modified nucleotides, and the modified nucleotides are selected from at least one of nucleotides having non-natural bases and nucleotides having natural modified bases; the non-natural bases include at least one of X, Y, and Z; the natural modified bases include at least one of Um, Ψ, m1Ψ, m5Um, m1G, Gm, m1A, Cm, m5C, and m3C.

3. The engineered tRNA for site-directed modification according to claim 2, characterized in that: The tRNA is selected from tRNA carrying leucine (Leu), and the modification of the tRNA molecule is to replace one or more sites simultaneously: the nucleotide at position 34 is replaced by a 2'-O-methylated modified nucleotide such as Cm and Um, the nucleotide at position 9 is replaced by m1G, the nucleotide at position 17 is replaced by Gm, the nucleotide at position 18 is replaced by Gm, the nucleotide at position 32 is replaced by Cm, the nucleotide at position 35 is replaced by Ψ, the nucleotide at position 37 is replaced by m1G, the nucleotide at position 38 is replaced by Ψ, the nucleotide at position 39 is replaced by Ψ or Um, the nucleotide at position 54 is replaced by Um or m5Um, the nucleotide at position 55 is replaced by Ψ or Um, the nucleotides at positions 17 and 34 are replaced by Gm and Cm, the nucleotides at positions 17 and 55 are replaced by Gm and Ψ, the nucleotides at positions 32 and 38 are replaced by Cm and Ψ, and the nucleotides at positions 34 and 55 are replaced by Cm and Ψ; or The tRNA is a tRNA carrying glutamine (Gln), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 9 with m1G, replacing the nucleotide at position 49 with m5C, replacing the nucleotide at position 39 with Um or Ψ, replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with a 2'-O-methylated modified nucleotide such as Cm and Um, replacing the nucleotides at positions 18 and 34 with Gm and Cm, respectively, replacing the nucleotide at position 4 with Um, replacing the nucleotide at position 13 with Ψ, and replacing the nucleotide at position 14 with is m1A, the nucleotide at position 18 is substituted with Gm, the nucleotide at position 27 is substituted with m5C, the nucleotide at position 28 is substituted with Ψ, the nucleotide at position 32 is substituted with Cm, the nucleotide at position 35 is substituted with Ψ, the nucleotide at position 36 is substituted with Z, the nucleotide at position 37 is substituted with a non-natural base X or Y, the nucleotide at position 39 is substituted with Ψ or Um, the nucleotide at position 54 is substituted with Um or m5Um, the nucleotide at position 55 is substituted with Ψ or Um, the nucleotide at position 58 is substituted with m1A, the nucleotide at position 72 is substituted with m5C, and the nucleotides at positions 18 and 55 are substituted with Gm and Ψ; or The tRNA is a tRNA carrying serine (Ser), and the tRNA molecule comprises one or more modifications selected from the following: replacing the nucleotide at position 18 with Gm, replacing the nucleotide at position 34 with Cm or Um, and replacing the nucleotide at position 35 with Ψ.

4. Use of the site-directed modified engineered tRNA according to any one of claims 1 to 3 in correcting nonsense mutations caused by premature termination codons (PTCs).

5. An engineered tRNA for analyzing in vivo distribution, which is obtained by modifying the site-specifically modified engineered tRNA according to any one of claims 1 to 3 with Cy5.

6. An engineered tRNA for delivery, which is the engineered tRNA for site-directed modification according to any one of claims 1 to 3 modified by GalNac.

7. Use of the site-directed modified engineered tRNA according to any one of claims 1 to 3 in the preparation of a drug for treating nonsense mutation diseases, wherein: The drug is an engineered tRNA delivered by LNP.

8. The use according to claim 7, characterized in that The LNP delivery is SM102 delivery.

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