Detection of markers in nascent proteins

Abstract

The invention is directed to methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are misaminoacylated with non-radioactive markers which may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. Markers may comprise cleavable moieties, detectable labels, reporter properties wherein markers incorporated into protein can be distinguished from unincorporated markers, or coupling agents which facilitate the detection and isolation of nascent,protein from other components of the translation system. The invention also comprises proteins prepared using misaminoacylated tRNAs which can be utilized in pharmaceutical compositions for the treatment of diseases and disorders in humans and other mammals, and kits which may be used for the detection of diseases and disorders.

1 . A conjugate, comprising a substrate covalently coupled to a detectable moiety through a cleavable linker, said substrate selected from the group consisting of nucleosides and nucleotides, said detectable moiety having the property of fluorescence.  
     
     
         2 . The conjugate of  claim 1 , wherein said substrate is a nucleotide selected from the group consisting of NTP, dNTP and ddNTP.  
     
     
         3 . The conjugate of  claim 2 , wherein said nucleotide is part of an oligonucleotide.  
     
     
         4 . The conjugate of  claim 1 , wherein said detectable moiety comprises coumarin.  
     
     
         5 . The conjugate of  claim 1 , wherein said detectable moiety is selected from the group consisting of rhodamines and fluoresceins.  
     
     
         6 . A method, comprising: 
 a) providing i) nucleic acid, ii) the conjugate of  claim 1 , and iii) a polymerase; and    b) mixing said nucleic acid and said polymerase in the presence of said conjugate under conditions such that said conjugate is incorporated into said nucleic acid to produce labeled nucleic acid.    
     
     
         7 . The method of  claim 6 , wherein said nucleic acid is RNA.  
     
     
         8 . The method of  claim 6 , wherein said nucleic acid is DNA.