Site-specifically-modified engineered trna and use thereof

Abstract

A site-specifically-modified engineered tRNA and the use thereof. Said modification refers to replacement, with one or more modified nucleotides, of one or more native unmodified nucleotides selected from sites 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58 and 72 of a tRNA molecule. Compared with unmodified tRNAs, the site-specifically-modified tRNA has higher structural stability and / or higher nuclease degradation resistance, the aminoacylation efficiency thereof is not remarkably reduced, and immunogenicity thereof is not remarkably increased. The modified tRNA can be used for eliminating premature translation terminations caused by nonsense mutations, and thus has great application potentials in treating nonsense mutation-related genetic diseases, tumors, etc. The modification of the engineered tRNA also provides valuable insights for drug development of tRNAs and thus can guide the modification design of native tRNAs.

Description

A site-modified engineered tRNA and its applications Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a site-modified engineered tRNA and its applications, and more particularly to a method for improving structural stability and decoding ability by modifying engineered tRNA. Background Technology

[0002] For a long time, scientists have followed the flow of genetic information in the central dogma to develop drugs, resulting in a plethora of drugs targeting DNA, RNA, and proteins. In particular, the outbreak of the COVID-19 pandemic at the end of 2019 propelled mRNA technology to new heights of interest. However, at the same time, many limitations have gradually emerged, such as high technical barriers, numerous patent restrictions, and an immature industrial chain, which have made it difficult to achieve breakthroughs in the short term and thus limited its development.

[0003] tRNAs were once considered abundant, widespread, and passively involved mRNA decoders and protein translation elements. By binding their anticodons to mRNA codons, tRNAs transport specific amino acids to complete protein translation. tRNAs carrying the same amino acid but with different anticodons are called "isoacceptors"; tRNAs carrying the same amino acid and the same anticodon but with different sequences are called "isodecoders." Recent research has unexpectedly discovered that tRNA isoacceptors and isodecoders possess specific, non-passively involved, active regulatory functions in translation, significantly influencing biological processes and disease progression. Consequently, scientists have gradually shifted their focus from mRNA to next-generation RNA technologies, exploring their potential, and tRNAs, previously overlooked by the industry, have emerged as a promising candidate.

[0004] tRNA, short for transfer RNA, is part of the cellular protein synthesis mechanism. tRNA "reads" the three-nucleotide codons on mRNA and then transports the corresponding amino acids to the ribosomes, where they are added to the continuously generated polypeptide chain. This process continues until the ribosome encounters a stop codon, at which point the ribosome detaches from the mRNA, completing protein synthesis. However, in some cases, gene mutations can change the codon encoding the amino acid into a stop codon, leading to premature termination of protein synthesis and loss of protein function. It is estimated that approximately 11% of genetic diseases are caused by gene mutations that result in premature termination of protein translation due to early stop codon formation (i.e., nonsense mutations).

[0005] The human genome contains 61 codons that can be recognized by tRNAs, encoding 20 amino acids. Three stop codons (UAG, UAA, and UGA) lack corresponding tRNA recognition, do not encode amino acids, and thus terminate translation. However, research has found tRNAs that can recognize stop codons, enabling them to encode amino acids and allowing protein translation to proceed normally. These tRNAs capable of recognizing stop codons are called nonsense-mutated engineered tRNAs. Engineered tRNAs are widely found in both plant and animal cells. Engineered tRNAs can overcome premature termination codons (PTCs). When they bind to PTCs, they do not cause protein synthesis to stop; instead, they add the correct amino acid at the PTC position, allowing protein synthesis to complete successfully. Therefore, theoretically, for genetic diseases caused by PTCs, only one engineered tRNA is needed to eliminate the premature translation interruption 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 using engineered tRNAs. In the early 21st century, researchers at the University of Colorado Boulder validated the feasibility of this therapy in transgenic mice. Subsequently, researchers at the Ott Institute of Obstetrics and Gynecology in St. Petersburg, Russia, used engineered tRNAs to correct the disease phenotype in a mouse model of Duchenne muscular dystrophy. In 2014, researchers in Portugal demonstrated that engineered tRNAs have therapeutic and preventative potential for hereditary tumor syndromes caused by nonsense mutations.

[0007] Currently, tRNA therapy is still in the exploratory stage, but signs of industrialization are gradually emerging. Companies such as Shape Therapeutics, Tevard Biosciences, ReCode Therapeutics, Alltrna, hC Bioscience, and Qixia Decoding Biotechnology are all conducting research on the development of therapeutic tRNAs, targeting diseases including Rett syndrome, hereditary epilepsy, Dravet syndrome, and Duchenne muscular dystrophy.

[0008] The number of nucleotides in tRNA falls between that of siRNA and mRNA, therefore its molecular weight is generally larger than that of ASO, siRNA, Aptamer, and gRNA, but smaller than that of mRNA. Because the complex stem-loop structure of tRNA is more similar to that of aptamers, their diameters are also similar. This suggests that tRNA can leverage drug development strategies from other nucleic acid drugs (Figure 1). There are many related studies on the modification and delivery of siRNA and mRNA.1

[0009] Although existing research shows that tRNA has promising prospects for industrial applications, practical applications still face challenges such as low decoding efficiency for premature stop codons (PTCs), poor stability, and unverified safety. Therefore, there is an urgent need to improve the structural stability of tRNA, its resistance to nuclease degradation, reduce immunogenicity, and maintain aminoacylation efficiency. However, the low levels of naturally occurring endogenous engineered tRNAs in vivo and the unclear factors affecting PTC decoding efficiency pose significant difficulties for tRNA structural modification. Summary of the Invention

[0010] To address the technical problems of low decoding efficiency, poor stability, and unverifiable safety of tRNA for premature stop codons (PTCs) in existing technologies, the present invention aims to provide a site-modified engineered tRNA. The modification involves replacing one or more naturally occurring 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 or naturally occurring modified bases. The site-modified tRNA can carry natural amino acids and recognize premature stop codons (PTCs). Compared with unmodified tRNA, the site-modified tRNA of the present invention exhibits higher structural stability and / or stronger resistance to nuclease degradation, and its aminoacylation efficiency is not significantly reduced, nor is its immunogenicity significantly increased. The modified tRNA can be used to eliminate premature translation termination caused by nonsense mutations, thus showing great application potential in the treatment of nonsense mutation-related genetic diseases and tumors. tRNA modification also provides insights for tRNA drug development, guiding the design of modifications to natural tRNAs.

[0011] Invention Summary

[0012] On the one hand, the present invention provides a site-modified engineered tRNA, wherein the modification includes 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 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.

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

[0015] Furthermore, the site-modified engineered tRNA of the present invention further includes modifications selected from one or more of the following sites: 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 at one or more sites involves replacing the natural nucleotide at one or more sites with the modified nucleotide.

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

[0017] Furthermore, the engineered tRNA with site-directed modification described in this invention is a tRNA carrying at least one of the following amino acids: glutamine (Gln), tryptophan (Trp), tyrosine (Tyr), and leucine (Leu).

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

[0019] Furthermore, the engineered tRNA with site-directed modification described in this invention is selected from tRNAs carrying tyrosine residues (Tyr), and the modification of the tRNA molecule involves simultaneously replacing one or more sites: the 34th nucleotide is replaced with a 2'-O-methylated nucleotide such as Cm and Um, the 18th nucleotide is replaced with Gm, the 32nd nucleotide is replaced with Cm, the 35th nucleotide is replaced with Ψ, the 37th nucleotide is replaced with m1G, the 39th nucleotide is replaced with Ψ or Um, the 54th nucleotide is replaced with Um or m5Um, the 55th nucleotide is replaced with Ψ or Um, the 58th nucleotide is replaced with m1A, the 18th and 34th nucleotides are replaced with Gm and Cm, and the 18th and 55th nucleotides are replaced with Gm and Ψ.

[0020] Furthermore, the engineered tRNA with site-directed modification described in this invention is selected from tRNAs carrying leucine (Leu), and the modification of the tRNA molecule involves simultaneously replacing one or more sites: the 34th nucleotide is replaced with a 2'-O-methylated nucleotide such as Cm and Um; the 9th nucleotide is replaced with m1G; the 17th nucleotide is replaced with Gm; the 18th nucleotide is replaced with Gm; the 32nd nucleotide is replaced with Cm; the 35th nucleotide is replaced with Ψ; the 37th nucleotide is replaced with m1G; the 38th nucleotide is replaced with Ψ; the 39th nucleotide is replaced with Ψ or Um; the 54th nucleotide is replaced with Um or m5Um; the 55th nucleotide is replaced with Ψ or Um; the 17th and 34th nucleotides are replaced with Gm and Cm; and the 17th and 55th nucleotides are replaced with Gm. The nucleotides at positions 32 and 38 are replaced with Cm and Ψ, and the nucleotides at positions 34 and 55 are replaced with Cm and Ψ.

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

[0022] Furthermore, the engineered tRNA with site-directed modification described in this invention includes modifications at the following two sites: replacing the 18th nucleotide with Gm and replacing the 34th nucleotide with a 2'-O-methylated nucleotide, such as Cm / Um.

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

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

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

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

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

[0028] On the other hand, the present invention provides a method for improving tRNA translation activity and / or enhancing tRNA decoding efficiency, wherein at least the 34th nucleotide of the tRNA is replaced with a 2'-O-methylated nucleotide; wherein 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 invention also provides the use of site-directed modified engineered tRNAs in correcting nonsense mutations caused by premature stop codons (PTCs).

[0029] Invention Details

[0030] On the one hand, the present invention provides a site-modified engineered tRNA, wherein the modification includes 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 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.

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

[0033] The nucleotide numbering positions on the tRNA molecule described in this invention are well known in the art, specifically referring to the nucleotide numbering positions shown in Figure 4a. Although the sequence lengths of tRNAs in the prior art are not entirely the same, they all have the secondary structure shown in Figure 4a, and those skilled in the art can determine the nucleotide sites of the tRNA based on the nucleotide numbering positions corresponding to those in Figure 4a.

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

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

[0036] Preferably, in the site-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-modified engineered tRNA of the present invention is a tRNA carrying at least one of the following amino acids: glutamine (Gln), tryptophan (Trp), tyrosine (Tyr), leucine (Leu), serine (Ser), lysine (Lys), glutamic acid (Glu), arginine (Arg), cysteine ​​(Cys), and glycine (Gly).

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

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

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

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

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

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

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

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

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

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

[0048] On the other hand, the present invention also provides a method for improving tRNA translation activity and / or enhancing tRNA decoding efficiency, wherein at least the 34th nucleotide of the tRNA is replaced with a 2'-O-methylated nucleotide; wherein 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] The tRNA molecule is modified at one or more sites selected from the following group: positions 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58, and 72; the modification of the one or more sites involves replacing the natural nucleotide at one or more sites with the modified nucleotide;

[0051] The modified nucleotide is selected from at least one of nucleotides having non-natural bases and nucleotides having naturally modified bases; the non-natural bases include at least one of X, Y, and Z; the naturally modified bases include 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] This invention first draws upon the modification paradigm of natural tRNA, combining 3D simulation results of tRNA molecular structure to select modification sites, evaluate the impact of each modification site on tRNA performance, and initially screen out modification sites and modification patterns that are beneficial to tRNA performance. tRNAs carrying four amino acids are then chemically synthesized to verify the impact of the initially screened beneficial modification sites and modification patterns on tRNA translation activity. The modification sites and modification patterns most beneficial to translation activity are selected to further verify their decoding efficiency for PTC.

[0054] First, this invention demonstrates that naturally occurring tRNA modification sites and paradigms are not always beneficial for structural stability, tRNA translation activity, and PTC decoding and readout efficiency. Simply considering structural stability 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 comprehensively.

[0055] Secondly, this invention has found that although tRNA molecules carrying different amino acids produce different effects when modified at the same site, the 34-position Cm modification exhibits better structural stability, higher translation activity, and stronger readability in both tRNAs carrying different amino acids and tRNA heterologous decoding family molecules carrying the same amino acid.

[0056] Third, this invention uses the GFP mRNA of the 39-position UAG from previous studies as a reporter system to study the function of modified tRNA, which more realistically reflects the biological function of tRNA in cells. The research method is simple and quick, and the results are more realistic and accurate. Attached Figure Description

[0057] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0058] Figure 1: Comparison of diameter and molecular weight of commonly used nucleic acid therapy drugs.

[0059] Figure 2: Modification sites of tRNA and modified nucleotides.

[0060] Figure 3: In tRNA Gln or tRNA Trp All or random modified nucleotides are introduced, wherein:

[0061] (a) Ψ, m1Ψ, Z-tRNAs with different proportions of modified nucleotides incorporated into them 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: Chemically synthesized site-specific modified tRNA Gln tRNA Tyr tRNA Leu and tRNA Trp ,in:

[0065] Figure 4a: This table summarizes the considerations for introducing tRNA modifications and the sites where modifications are introduced. In the table, red text represents natural endogenous modifications involved in this experiment; blue text represents artificial or non-natural base modifications in the nucleic acid drugs involved in this experiment.

[0066] Figure 4b: tRNA Gln Fluorescence image 24 h after co-transfection of HEK293T with GFP-UAG mRNA;

[0067] Figure 4c: tRNA Trp Fluorescence image 24 h after co-transfection of HEK293T with GFP-UAG mRNA;

[0068] Figure 4d: tRNA Tyr Fluorescence image 24 h after co-transfection of HEK293T with GFP-UAG mRNA;

[0069] Figure 4e: tRNA Leu Fluorescence image 24 h after co-transfection of HEK293T with GFP-UAG mRNA.

[0070] Figure 5: Chemically synthesized tRNA with site-directed modification that reads through three nonsense mutant codons, wherein:

[0071] (a) Modified tRNA Ser(CTA) tRNA Lys(CTA) tRNA Glu(CTA) Fluorescence image 24 h after co-transfection of HEK293T with GFP-UAG mRNA;

[0072] (b) Modified tRNA Gln(TTA) tRNA Leu(TTA) tRNA Tyr(TTA) tRNA Ser(TTA) Fluorescence image 24 h after co-transfection of HEK293T with GFP-UAA mRNA;

[0073] (c) Modified tRNA Arg(TCA) tRNA Gly(TCA) tRNA Cys(TCA) tRNA Leu(TCA) tRNA Ser(TCA) Fluorescence image 24 h after co-transfection of HEK293T with GFP-UGA mRNA;

[0074] (d) tRNA Gln(CTA) Fluorescence and flow cytometry analysis of isodecoders after 2'-O-methylation at position 34, co-transfected with GFP-UAG mRNA into HEK293T at 24-hour 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 34-position 2'-O-methylation modification, the tRNA was co-transfected with GFP-UAG / UAA / UGA mRNA into HEK293T and then analyzed by flow cytometry. Figure 6: Characterization and application of chemically synthesized tRNAs with 34Cm modification, Cy5 modification, and GalNac.

[0076] In Figure 6: (a) tRNA 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 transfection of MPS IH nonsense mutant mice (B6.129S-Idua) tm1.1Kmke / Fibroblasts of J). The IDUA gene of this fibroblast contains a TAG mutation at position 392;

[0077] (b) In tRNA Gln(CTA) The fluorescence image of Cy5-labeled 5', 3' ends and D-loop of -34Cm mRNA after co-transfection with GFP-UAG mRNA into HEK293T cells for 24 hours. Labeled as: 5'-Cy5-Gln(CTA)-34Cm, 3'-Cy5-Gln(CTA)-34Cm, D-Cy5-Gln(CTA)-34Cm;

[0078] (c) Modify the 3'-Cy5-tRNA of GalNac at the 5' and 3' ends. Gln(CTA) -34Cm or D-Cy5-tRNA Gln(CTA) Fluorescence image of HEK293T cells 24 h after co-transfection with 34Cm and GFP-UAG mRNA. Labeled as: 5'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: 5'-modified GalNac D-Cy5-Gln(CTA)-34Cm, 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: 3' end modified GalNac D-Cy5-Gln(CTA)-34Cm, 5'-GalNac-3'-Cy5-tRNA Gln(CTA) -34Cm: 5' end modified GalNac 3'-Cy5-Gln(CTA)-34Cm;

[0079] (d) Using 0.9% saline as a control, D-Cy5-tRNA was delivered using Polyplus (catalog number: 101000040) and LNP (Lipid Nanoparticle, SM102), respectively. Gln(CTA) -34 cm, tail vein injection (IV), intramuscular injection (IM), intraperitoneal injection (IP), and comparison of tRNA distribution;

[0080] (e) Using 0.9% saline as a control, D-Cy5-tRNA was delivered using LNP. Gln(CTA) and D-Cy5-tRNA Gln(CTA)-34 cm, administered via tail vein injection (IV) to mice. Distribution time before and after modification was compared;

[0081] (f) Subcutaneous injection of 5'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm and 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34 cm. Detailed Implementation

[0082] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0083] Non-natural base pairs are crucial building blocks for synthetic life. Life on Earth evolved from a common ancestor and shares a genetic code. This biological information is stored in DNA, composed of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and deoxythymidine (dT), stored and retrieved by forming two base pairs: (d)A-dT / U and (d)G-(d)C. Scientists have developed non-natural base pairs beyond the four base pairs (UBP), injecting new elements into the storage and transmission of genetic information and facilitating the processes of DNA replication, RNA transcription, and translation. This study used the dNaM(dX)-dTPT3(dY) and NaM(X)-TPT3(Y) base pairs to introduce the non-natural base NaM(X) or TPT3(Y) into the 37th position of tRNA. 9 Due to the resistance of non-natural bases to endogenous nucleases, it is desirable to enhance the stability of engineered tRNAs. 2'-aminoadenine (Z) is also a non-natural base, found in Bacteriophage. 2 Z has one more amino group than natural adenine, which theoretically enhances the interaction between bases. Zhang M et al. reported that incorporating Z into mRNA resulted in Z-mRNA with higher translation efficiency, lower cytotoxicity, and lower immunogenicity compared to unmodified mRNA. 3Pseudouridine (Ψ), Methylguanosine (Gm), 2'-O-Methylcytidine (Cm), 2'-O-Methyluridine (Um), 5-Methyluridine (m5U), 3-Methylcytidine (m3C), 1-Methyladenosine (m1A), 5-Methylcytidine (m5C), and 1-Methylguanosine (m1G) are also potential candidate tRNA modifications. Ψ, as a commonly used modification in mRNA vaccines, can reduce mRNA immunogenicity. 4 Similarly, introducing Ψ into tRNA is expected to reduce the immunogenicity of exogenously introduced tRNA. However, if Ψ is completely replaced by U in mRNA, the higher-order structure of tRNA may be disrupted, leading to its loss of function. Gm, Cm, and Um are common ribose 2'-O-methylation modifications, commonly found in siRNA modifications, which help resist nucleases and reduce immune stimulation to some extent. These modifications are also common in endogenous tRNA and are related to immune recognition and decoding efficiency. m5U, m3C, m1A, m5C, and m1G are common modifications of endogenous tRNA. 5 m5C, m5U, m1A 4,6 These are also modified nucleotides that have been screened for on mRNA vaccines. This suggests that these modifications are promising potential candidates.

[0084] The modifications in this invention mainly involve non-natural bases X, Y, and Z, common nucleic acid drug modifications, and endogenous RNA modifications Um, Ψ, m1Ψ, m5U, m1G, Gm, m1A, Cm, m5C, and m3C. The nucleotide modifications on tRNA involved in this invention are shown in Figure 2. More than one modifying nucleotide is designed at positions 34, 37, 39, 54, and 55, and their structural formulas are listed. The introduction of modifying nucleotides follows the principles of enhancing tRNA decoding activity, improving stability, and reducing immune stimulation. Potential modifying nucleotides are selected from non-natural base pairs in synthetic biology, modifying nucleotides of nucleic acid drugs, and modifying nucleotides of natural tRNA itself, thus screening for ideal modification paradigms. Furthermore, considering the effectiveness and safety issues in practical applications, some modifications, while not enhancing tRNA translation activity, may potentially reduce its immune activity and increase nuclease resistance. Therefore, if the modified tRNA possesses translational activity, it should be considered a beneficial modification.

[0085] Example 1: Introducing non-natural bases into in vitro transcribed IVT-tRNA

[0086] Based on previous research, GFP mRNA containing UAG was used as a reporter system. 7 When preparing tRNA, referencing the preparation of in vitro transcribed mRNA, T7-tRNA was selected. Gln As a transcription template, all U or A in the in vitro transcription system are replaced with Ψ. 4 , m 1 Ψ 8 and Z 3 (Figure 3(a)) revealed that the Ψ, m1Ψ, Z-tRNA obtained from in vitro transcription was... Gln Decoding activity is low. (Using Z-tRNA) Gln For example, by gradually changing the incorporation ratio of Z, the ratio of A:Z was gradually increased 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.

[0087] In addition, we attempted to introduce non-natural bases into tRNA using non-natural base pairs (Figure 3(b)).

[0088] We selected the dNaM(dX)-dTPT3(dY) and NaM(X)-TPT3(Y) base pairs. 9 Given the hydrophobicity of X and Y, they were chosen for insertion into the tRNA. Gln and tRNA Trp Position 37. This position is adjacent to the anticodon, and modification at this site helps ensure the mRNA... 10,11 Accurate decoding and maintaining the correct reading frame are crucial for preserving the anticodon ring structure. 12,13 This promotes the interaction between tRNA and mRNA with rRNA. The hydrophobic structure modified with purine at position 37 theoretically helps disrupt the 33U-37A interaction, forming a U-turn in the anticodon loop. 12–14 37X-tRNA Gln The expression of GFP-UAG was restored, providing an initial structure for subsequent optimization.

[0089] Example 2: In chemically synthesized tRNA Gln tRNA Tyr tRNA Leu tRNA Trp Introducing endogenous modifications

[0090] Full-length tRNA can be synthesized in a solid-phase manner using phosphoramide monomers, with modified nucleotides incorporated at specific sites. This method enables high-throughput screening of beneficial modification sites. The modifications are introduced to reduce the immunogenicity of the tRNA. 16–18 This enhances its nuclease resistance, thereby prolonging its action time, and serves as a recognition element for aminoacyl-tRNA synthetase.19,20 Stabilize tRNA structure 12 Promotes the binding of metal ions 11 Fine-tuning the tRNA structure promotes the interaction between mRNA and rRNA to enhance decoding efficiency. 13 .

[0091] Here, based on our understanding of existing tRNA modifications, we employ an engineered approach to first screen for single-site modifications and then further evaluate two-site modifications, providing methods and insights for subsequent mechanistic studies and optimization of drug efficacy. We combine this with an analysis of the endogenous modification profile of tRNA. 5 and the functions of each part of the tRNA structure, the relationship between tRNA modification and tRF. 25 siRNA modification substitution was used to map the possible strategies for introducing tRNA modification (Figure 4a).

[0092] For tRNA Gln Generally speaking, modifications such as 9m1G, 18Gm, 34Cm, 39Ψ, 39Um, 49m5C, 54Um, 55Ψ, and double-site 2'-O-methylation substitutions at positions 18 and 34 can all enhance translational activity. 18Gm is a crucial modification distinguishing prokaryotic and eukaryotic tRNAs; bacterial tRNAs modified with 18Gm can evade immunity. 17 34 cm may slightly reduce immune stimulation. 28,29 It resists nuclease cleavage. Theoretically, T / D-loop modification can help tRNA form a more stable tertiary structure. 12 .

[0093] For tRNA Trp For example, unmodified tRNA Trp The translation activity was low, but modifications at 17Gm, 18Gm, 34Cm, 35Ψ, 37m1G, 54m5Um, 55Ψ, 17Gm+34Cm, 18Gm+34Cm, and 34Cm+55Ψ increased its translation activity (Figure 4c). Modification at both 34Cm and 35Ψ sites greatly enhanced the translation activity.

[0094] For tRNA Tyr For example, 34Cm, 35Ψ, and 58m1A enhance its translation activity (Figure 4d).

[0095] For tRNA Leu For example, 34Cm, 35Ψ, 38Ψ, 55Ψ, and 17Gm+34Cm enhance its translation activity (Figure 4e).

[0096] Example 3: 2'-O-methylation at position 34 affects tRNA decoding efficiency.

[0097] Besides the four amino acids mentioned above, the modification paradigm is similar for tRNAs corresponding to other amino acids.

[0098] Figures 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) tRNA Ser(TCA) Changes in translational activity before and after modification. tRNA Arg(TCA) It is relatively sensitive to modifications; 55Ψ does not impair its translational activity (Figure 5(c)). tRNA Ser(CTA) The 18 Gm value does not affect its translation activity, but 34 Cm and 35 Ψ effectively enhance its translation activity (Figure 5(a)).

[0099] Figure 6 shows 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 following 2'-O-methylation at position 34. tRNA Ser(CTA) tRNA Glu(CTA) tRNA Leu(TCA) tRNA Ser(TCA) tRNA Leu(UUA) The 34-position 2'-O-methylation modification can significantly enhance its translation activity (Figure 5(e)).

[0100] In addition, tRNA Gln-CTGThe 2'-O-methylation modification at position 34 of the six tRNAs that are themselves translationally active can significantly improve their translation efficiency (Figure 5(d)).

[0101] Example 4: Cy5 labeling can trace tRNA distribution, and GalNac modification facilitates tRNA delivery.

[0102] To further investigate the translational activity and possible mechanism of 2'-O-methylation modification, B6.129S-Idua was prepared. tm1.1Kmke / Fibroblasts from mice with J nonsense mutation disease were transfected with tRNAs before and after 2'-O-methylation at position 34. Tyr(CTA) tRNA Ser(CTA) tRNA Gln(CTA) tRNA Leu(CTA) Protein expression differences were analyzed after 24 hours (Figure 6(a)). We found that compared with the control group, the IDUA protein expression level in the tRNA-transfected experimental group was significantly increased, with tRNA showing the highest expression level. Tyr(CTA) The improvement was more significant after modification with a 2'-oxymethyl group at position 34.

[0103] In tRNA Gln(CTA) The 3', 5', and 16-position D-loop of the -34Cm RNA were labeled with Cy5 dye, denoted as: 5'-Cy5-Gln(CTA)-34Cm, 3'-Cy5-Gln(CTA)-34Cm, and D-Cy5-Gln(CTA)-34Cm. The translational activity of these three markers was verified (Figure 6(b)). In the 3'-Cy5-tRNA... Gln(CTA) -34Cm or D-Cy5-tRNA Gln(CTA) The 5' and 3' ends of the 5' and 3' ends of the 5'-Cy5-tRNA are modified. This is denoted as: 5'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: 5'-modified GalNac D-Cy5-Gln(CTA)-34Cm, 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm: 3' end modified GalNac D-Cy5-Gln(CTA)-34Cm, 5'-GalNac-3'-Cy5-tRNA Gln(CTA) -34Cm: 5' end modified GalNac 3'-Cy5-Gln(CTA)-34Cm. Verification showed that all three had translational activity (Figure 6(c)).

[0104] Using 0.9% saline as a control, D-Cy5-Gln(CTA)-34Cm was delivered via Polyplus and LNP. The distribution and residence of tRNA in mice could be reflected by detecting Cy5 signaling (Figure 6(d)). tRNA delivered via intravenous injection using LNP... Gln(CTA) -34 cm compared to unmodified tRNA Gln(CTA) The tRNA has a longer residence time in mice (Figure 6(e)). This indicates that the 2'-O-methylation modification at position 34 can enhance tRNA stability, prolong its duration of action, and thus enhance its translational activity.

[0105] Subcutaneous injection of 5'-GalNac-D-Cy5-tRNA Gln(CTA) -34Cm and 3'-GalNac-D-Cy5-tRNA Gln(CTA) -34 cm, 12 h later, 5' GalNac-modified tRNA was observed to accumulate in the peritoneal cavity, which can directly deliver GalNac-modified tRNA to treat nonsense mutation diseases.

[0106] By drawing inspiration from natural tRNA modifications, we can optimize the modification and delivery of tRNA drugs. Future research will further optimize tRNA modification paradigms, such as exploring the introduction of more non-endogenous modifications to enhance their anti-nuclease properties and prolong their duration of action in vivo without significantly impacting tRNA translational activity.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. The patent protection scope of the present invention should be determined by the described claims.

[0108] The sequence structure of the modified tRNA synthesized in this invention is as follows:

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] The following prior art is incorporated herein by reference in its entirety:

[0117]

[0118]

[0119] .

Claims

1. A site-modified engineered tRNA, characterized in that: The modification includes replacing at least the 34th position of the natural nucleotide in the tRNA molecule with a modified nucleotide; The modified nucleotide is 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. The site-modified tRNA can carry natural amino acids and recognize premature stop codons (PTCs).

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

3. The engineered tRNA with site-directed modification as described in claim 1 or 2, characterized in that, 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.

4. The engineered tRNA with site-directed modification as described in claim 1 or 2, characterized in that: The tRNA mentioned is a tRNA carrying at least one of the following amino acids: glutamine (Gln), tryptophan (Trp), tyrosine (Tyr), leucine (Leu), serine (Ser), lysine (Lys), glutamic acid (Glu), arginine (Arg), cysteine ​​(Cys), and glycine (Gly).

5. The engineered tRNA with site-directed modification as described in claim 4, characterized in that: The tRNA is a tRNA carrying tryptophan (Trp), and the tRNA molecule contains modifications selected from one or more of the following: replacing the 34th nucleotide with a 2'-O-methylated nucleotide such as Cm and Um; replacing the 35th nucleotide with Ψ; replacing the 34th and 35th nucleotides with Cm and Ψ; replacing the 9th nucleotide with m1G; replacing the 17th nucleotide with Gm; replacing the 18th nucleotide with Gm; replacing the 32nd nucleotide with m3C; replacing the 36th nucleotide with Z; replacing the 37th nucleotide with m1G or X and Y; replacing the 39th nucleotide with Ψ or Um; replacing the 54th nucleotide with Um or m5Um; replacing the 55th nucleotide with Ψ or Um; replacing the 34th nucleotide with Cm; replacing the 34th nucleotide with Cm; replacing the 17th and 34th nucleotides with Gm and Cm; replacing the 18th and 34th nucleotides with Gm. And Cm, the nucleotides at positions 34 and 55 are replaced with Cm and Ψ; or The tRNA is selected from tRNAs carrying tyrosine residues (Tyr), and the modification of the tRNA molecule involves the simultaneous replacement of one or more sites: the 34th nucleotide is replaced with a 2'-O-methylated nucleotide such as Cm or Um; the 18th nucleotide is replaced with Gm; the 32nd nucleotide is replaced with Cm; the 35th nucleotide is replaced with Ψ; the 37th nucleotide is replaced with m1G; the 39th nucleotide is replaced with Ψ or Um; the 54th nucleotide is replaced with Um or m5Um; the 55th nucleotide is replaced with Ψ or Um; the 58th nucleotide is replaced with m1A; the 18th and 34th nucleotides are replaced with Gm and Cm; and the 18th and 55th nucleotides are replaced with Gm and Ψ; or The tRNA is selected from tRNAs carrying leucine (Leu), and the modification of the tRNA molecule involves the simultaneous replacement of one or more sites: the 34th nucleotide is replaced with a 2'-O-methylated nucleotide such as Cm and Um; the 9th nucleotide is replaced with m1G; the 17th nucleotide is replaced with Gm; the 18th nucleotide is replaced with Gm; the 32nd nucleotide is replaced with Cm; the 35th nucleotide is replaced with Ψ; the 37th nucleotide is replaced with m1G; the 38th nucleotide is replaced with Ψ; the 39th nucleotide is replaced with Ψ or Um; the 54th nucleotide is replaced with Um or m5Um; the 55th nucleotide is replaced with Ψ or Um; the 17th and 34th nucleotides are replaced with Gm and Cm; the 17th and 55th nucleotides are replaced with Gm and Ψ; the 32nd and 38th nucleotides are replaced with Cm and Ψ; and the 34th and 55th nucleotides are replaced with Cm and Ψ; or The tRNA is a tRNA carrying glutamine (Gln), and the tRNA molecule contains modifications selected from one or more of the following: replacing the 9th nucleotide with m1G, replacing the 49th nucleotide with m5C, replacing the 39th nucleotide with Um or Ψ, replacing the 18th nucleotide with Gm, replacing the 34th nucleotide with a 2'-O-methylated nucleotide such as Cm or Um, replacing the 18th and 34th nucleotides with Gm and Cm respectively, replacing the 4th nucleotide with Um, replacing the 13th nucleotide with Ψ, and replacing the 14th nucleotide with... For m1A, the 18th nucleotide is replaced with Gm, the 27th nucleotide with m5C, the 28th nucleotide with Ψ, the 32nd nucleotide with Cm, the 35th nucleotide with Ψ, the 36th nucleotide with Z, the 37th nucleotide with a non-natural base X or Y, the 39th nucleotide with Ψ or Um, the 54th nucleotide with Um or m5Um, the 55th nucleotide with Ψ or Um, the 58th nucleotide with m1A, the 72nd nucleotide with m5C, and the 18th and 55th nucleotides with Gm and Ψ, respectively; or The tRNA is a tRNA carrying a serine residue (Ser), and the tRNA molecule contains one or more modifications selected from the following: replacing the 18th nucleotide with Gm, replacing the 34th nucleotide with Cm or Um, replacing the 35th nucleotide with Ψ; or The tRNA is a tRNA carrying a lysine residue (Lys), and the tRNA molecule contains one or more modifications selected from the following: replacing the 18th nucleotide with Gm, replacing the 34th nucleotide with Cm, replacing the 35th nucleotide with Ψ; or The tRNA is a tRNA carrying arginine (Arg), and the tRNA molecule contains one or more modifications selected from the following: replacing the 18th nucleotide with Gm, replacing the 34th nucleotide with Um or Ψ, replacing the 55th nucleotide with Ψ; or The engineered tRNA is a tRNA carrying glycine (Gly), and the tRNA molecule contains one or more modifications selected from the following: replacing the 18th nucleotide with Gm, replacing the 34th nucleotide with Um or Ψ, or replacing the 55th nucleotide with Ψ engineered tRNA; or The tRNA is a tRNA carrying a cysteine ​​residue (Cys), and the tRNA molecule contains one or more modifications selected from the following: replacing the 18th nucleotide with Gm, replacing the 34th nucleotide with Um or Ψ, and replacing the 55th nucleotide with Ψ.

6. Use of the site-modified engineered tRNA according to any one of claims 1-5 in correcting nonsense mutations caused by premature stop codons (PTCs).

7. A method for improving tRNA translation activity and / or enhancing tRNA decoding efficiency, characterized in that: At least the nucleotide at position 34 of the tRNA is replaced with a nucleotide modified by 2'-O-methylation; 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 At least one of the heterogeneous decoding family (isodecoder).

8. The method for improving tRNA translation activity and / or enhancing tRNA decoding efficiency as described in claim 7, comprising: The tRNA molecule is modified at one or more sites selected from the following group: positions 4, 9, 13, 14, 17, 18, 27, 28, 32, 34, 35, 36, 37, 38, 39, 49, 50, 54, 55, 58, and 72; the modification of the one or more sites involves replacing the natural nucleotide at one or more sites with the modified nucleotide; The modified nucleotide is selected from at least one of nucleotides having non-natural bases and nucleotides having naturally modified bases; the non-natural bases include at least one of X, Y, and Z; the naturally modified bases include at least one of Um, Ψ, m1Ψ, m5Um, m1G, Gm, m1A, Cm, m5C, and m3C.

9. An engineered tRNA for analyzing in vivo distribution, wherein the engineered tRNA of any one of claims 1-5 is modified with Cy5.

10. An engineered tRNA for delivery, wherein the engineered tRNA of any one of claims 1-5 is modified with GalNac.

11. The use of the site-modified engineered tRNA according to any one of claims 1-5 in the preparation of a drug for treating nonsense mutation diseases, wherein, The drug is an engineered tRNA delivered by LNP, preferably an engineered tRNA delivered by SM102.

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