Systems and methods for measuring translation of target proteins in cells

Abstract

The present invention relates to systems and methods for measuring the rate of translation of a target protein in cells, which are based on the detection of translation of one or more predetermined codon pairs during synthesis of the target protein. The detection is provided by a FRET signal emitted from labeled tRNA molecules which are juxtaposed during synthesis of the protein.

1 - 60 . (canceled) 
     
     
         61 . A tRNA FRET pair capable of reading at least one predetermined codon pair in an mRNA sequence encoding a protein of interest; and wherein one tRNA member of each tRNA pair is labeled with a donor fluorophore and the other tRNA member is labeled with an acceptor fluorophore. 
     
     
         62 . The tRNA FRET pair according to  claim 61 , wherein said at least one codon pair occurs in said mRNA sequence at a frequency that is at least five-fold higher than the general frequency of said at least one codon pair in a reference proteome or transcriptome. 
     
     
         63 . The tRNA FRET pair according to  claim 62 , wherein said at least one codon pair occurs in said mRNA sequence at a frequency that is at least ten-fold higher than the general frequency of said at least one codon pair in a reference proteome or transcriptome. 
     
     
         64 . The tRNA FRET pair according to  claim 61 , wherein the enrichment factor of said at least one codon pair is at least 6.0. 
     
     
         65 . The tRNA FRET pair according to  claim 61 , wherein each tRNA of said tRNA pair corresponds to one amino acid such that said each tRNA corresponds to a tRNA from the complete set of isoacceptor tRNAs specific for said one amino acid. 
     
     
         66 . The tRNA FRET pair according to  claim 61 , wherein said pair is selected from: Cysteine-Leucine, Tryptophan-Tryptophan, Cysteine-Tryptophan, Cysteine-Phenylalanine, Asparagine-Tryptophan, Tyrosine-Tryptophan, Arginine-Alanine, Proline-Arginine, Tyrosine-Serine, Arginine-Arginine, Alanine-Tyrosine, Arginine-Serine and Tyrosine-Phenylalanine. 
     
     
         67 . The tRNA FRET pair according to  claim 66 , wherein the pair Cysteine-Leucine is for measuring the translation of filgrastim;
 the pair Cysteine-Phenylalanine is for measuring the translation of somatotropin;   the pair is selected from Arginine-Alanine, Proline-Arginine and Tyrosine-Serine for measuring the translation of ALT1;   the pair is selected from Arginine-Arginine, Alanine-Tyrosine and Arginine-Serine for measuring the translation of ALT2; the pair is Aspargine-Tryptophan for measuring the translation of etanercept; and   the pair is Tyrosine-Tryptophan for measuring the translation of cetuximab.   
     
     
         68 . The tRNA FRET pair according to  claim 61 , wherein one tRNA member of said tRNA FRET pair is capable of reading synonymous codons encoding a particular amino acid. 
     
     
         69 . The tRNA FRET pair according to  claim 61 , wherein said tRNA pair corresponds to a specific pair of adjacent amino acids occurring at said protein of interest.