Read Through Of Truncated Proteins In Premature Termination Codon Diseases Using An Optimized Genetic Codon Expansion System

Abstract

Provided is a method for high-efficiently reading through a nonsense mutation site in a pathogenic gene in a monogenic hereditary disease and restoring the normal structure and function of a mutant protein, by using a genetic code expanded non-natural amino acid system. By modifying a tRNA of Methanosarcina barkeri (tRNAPyl), an all-new UAA and UGA encoded non-natural amino acid system that has high read-through efficiency is obtained, and the range of using the orthogonal pair of tRNAPyl and pyrrolysyl-tRNA synthetase (PylRS) is expanded. A plasmid mimicking the endogenous premature termination codon is constructed, so as to evaluate the efficiency of reading through the endogenous premature termination codon. Also provided is a system mainly comprising pathogenic genes of monogenic hereditary diseases and tumor inhibitory genes in tumor cells.

1 . A tRNA comprising a mutated anticodon loop, wherein the base CUA of the anticodon loop is mutated to UUA or UCA, and wherein, the mutated tRNA can be recognized by at least one non-natural aminoacyl tRNA synthetase which is orthogonal thereto. 
     
     
         2 . The tRNA of  claim 1 , wherein the anticodon loop of the tRNA is not bound to at least one non-natural aminoacyl tRNA synthetase which is orthogonal thereto. 
     
     
         3 . The tRNA of  claim 1 , wherein the tRNA is a tRNA derived from  Methanosarcina barkeri  (tRNAPyl), or a tRNA derived from  E. coli.    
     
     
         4 . The tRNA of  claim 1 , further comprising at least one non-natural aminoacyl tRNA synthetase which is orthogonal thereto or the encoding nucleic acid sequence thereof, thereby constituting a non-natural amino acid system. 
     
     
         5 . The non-natural amino acid system of  claim 4 , wherein the non-natural amino acid system is a lysine-like non-natural amino acid system, a leucine-like non-natural amino acid system, or a tyrosine-like non-natural amino acid system, wherein the lysine-like non-natural amino acid system comprises:
 Lys-diazirine (NAEK) of formula I   
       
         
           
           
               
               
           
         
       
       or
 Lys-azido of formula II 
 
       
         
           
           
               
               
           
         
       
       or
 wherein the leucine-like non-natural amino acid comprises Anap of formula III 
 
       
         
           
           
               
               
           
         
       
       or
 wherein the tyrosine-like non-natural amino acid comprises pAcF of formula IV 
 
       
         
           
           
               
               
           
         
       
     
     
         6 . The tRNA of  claim 1  in a plasmid, a vector, a host cell or a kit. 
     
     
         7 . A method for genetic codon expansion, wherein the base CUA of the anticodon loop of a tRNA is point-mutated to UUA or UCA, and wherein the mutated tRNA can be recognized by a non-natural amino acid tRNA synthetase orthogonal thereto. 
     
     
         8 . The method of  claim 7 , wherein the tRNA is a tRNA derived from  Methanosarcina barkeri  (tRNAPyl), or a tRNA derived from  E. coli.    
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . A method for restoring normal expression and function of a nonsense mutant protein, comprising introducing an effective amount of the non-natural amino acid system of  claim 4  to a cell or an organism comprising a nonsense mutant protein. 
     
     
         12 . The method of  claim 11 , wherein the introduced non-natural amino acid system recognizes a nonsense mutation of the protein of interest and introduces a non-natural amino acid at a corresponding site of the nonsense mutation to allow the translation of the protein of interest to avoid premature termination and to synthesize an intact functional protein. 
     
     
         13 . The method of  claim 12 , wherein the introduced non-natural amino acid is Lys-diazirine of formula I 
       
         
           
           
               
               
           
         
         Wherein R 1  is the amino acid immediately N-terminal to the non-natural amino acid, R 2  is the amino acid immediately C-terminal to the non-natural amino acid, and R 3  is 
       
       
         
           
           
               
               
           
         
       
       or Lys-azido of formula (II) 
       
         
           
           
               
               
           
         
         wherein R1 is the amino acid immediately N-terminal to the non-natural amino acid, R2 is the amino acid immediately C-terminal to the non-natural amino acid, and 
         R4 is 
       
       
         
           
           
               
               
           
         
       
     
     
         14 . A mammalian stable cell line HEK293-PYL, deposited on Nov. 17, 2015 under accession number CGMCC No. 11592. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 7 , wherein the mutated tRNA is tRNA Pyl   UUA  having the sequence of SEQ ID NO: 1, or tRNA Pyl   UCA  having the sequence of SEQ ID NO: 2. 
     
     
         18 . The method of  claim 9 , wherein the mutated tRNA is tRNA Pyl   UUA  having the sequence of SEQ ID NO: 1, or tRNA Pyl   UCA  having the sequence of SEQ ID NO: 2. 
     
     
         19 . The method of  claim 11 , wherein the mutated tRNA is tRNA Pyl   UUA  having the sequence of SEQ ID NO: 1, or tRNA Pyl   UCA  having the sequence of SEQ ID NO: 2. 
     
     
         20 . The method of  claim 9 , wherein the nonsense mutation is in the gene encoding the Dystrophin protein, the tumor suppressor gene STK11, or the gene encoding the EPHB2 protein. 
     
     
         21 . The method of  claim 11 , wherein the nonsense mutation is in the gene encoding the Dystrophin protein, the tumor suppressor gene STK11, or the gene encoding the EPHB2 protein. 
     
     
         22 . The method of  claim 11 , wherein the non-natural amino acid system of  claim 4  is introduced in a quantity effective to treat a hereditary disease or cancer caused by a nonsense mutation in a gene. 
     
     
         23 . The method of  claim 11 , wherein the wherein the hereditary disease or cancer is Duchenne muscular dystrophy, cystic fibrosis, hemophilia A, hemophilia B, lipid storage disease, ataxia telangiectasia, Hurler's syndrome, amaurotic familial idiocy, stomach cancer, or lung cancer.