Methods for producing nucleic acids lacking 3'-untranslated regions and optimizing cellular RNA -protein fusion formation

Abstract

Described herein are methods for removing the 3′-untranslated regions from cDNA or mRNA molecules, as well as methods for the use of such products for RNA-protein fusion formation.

What is claimed is:  
     
         1 . A library of nucleic acid molecules, each molecule comprising an open reading frame and lacking the 3′-untranslated region normally associated with said open reading frame.  
     
     
         2 . The library of  claim 1 , wherein said nucleic acid is RNA.  
     
     
         3 . The library of  claim 2 , wherein said RNA is messenger RNA.  
     
     
         4 . The library of  claim 2 , wherein said RNA is cellular RNA.  
     
     
         5 . The library of  claim 4 , wherein said cellular RNA is derived from a eukaryotic organism.  
     
     
         6 . The library of  claim 5 , wherein said cellular RNA is derived from a mammal.  
     
     
         7 . The library of  claim 6 , wherein said mammal is a human.  
     
     
         8 . The library of  claim 1 , wherein said nucleic acid is DNA.  
     
     
         9 . The library of  claim 1 , wherein said library comprises at least 10 5  members.  
     
     
         10 . The library of  claim 1 , wherein said nucleic acid molecules of said library also lack stop codons.  
     
     
         11 . A library of nucleic acid molecules produced by the steps of: 
 (a) providing a library of DNA molecules, each having an open reading frame and a 3′-untranslated region, each of said DNA molecules terminating at its 5′ end in an overhang and at its 3′ end in a blunt end; and    (b) treating said library of DNA molecules first with a 3′→5′ exonuclease and then with a single-stranded nuclease under conditions that allow removal of the 3′-untranslated regions of said DNA molecules.    
     
     
         12 . A library of nucleic acid molecules produced by the steps of: 
 (a) translating a library of mRNA molecules in vitro in a translation reaction mixture lacking functional translation release factor activity, resulting in pausing of the translation reaction mixture ribosomes at the stop codons of said mRNA molecules;    (b) adding, to said translation reaction mixture of step (a), reverse transcriptase and oligonucleotide primers which are complementary to the 3′-untranslated regions of said mRNA molecules at a site proximal to said stop codons, under conditions which allow the synthesis of strands of DNA that are complementary to said 3′-untranslated regions and terminate at sites proximal to said stop codons; and    (c) removing the RNA portions of the RNA-DNA duplexes formed in step (b), thereby removing the 3′-untranslated regions of said mRNA molecules.    
     
     
         13 . The library of  claim 12 , produced by the further steps of: 
 (d) ligating to each of the 3′ ends of the products of step (c) a linker comprising a Type IIS restriction site;    (e) extending the products of step (d) to produce double-stranded DNA molecules; and    (f) treating said double-stranded DNA molecules with a Type IIS restriction enzyme that recognizes said Type II restriction site to cleave said DNA molecules and remove said stop codons.    
     
     
         14 . A library of nucleic acid molecules produced by the steps of: 
 (a) providing a population of mRNA molecules;    (b) synthesizing strands of DNA, each of which is complementary to one of said mRNA molecules, using a random primer mixture, said random primer mixture comprising primers, each having 
 (i) a 3′ region comprising a stop codon flanked by a random oligonucleotide located 3′, 5′, or both to said stop codon; and  
 (ii) a 5′ region comprising a Type IIS restriction site;  
   (c) ligating to the 3′ ends of the DNA products of step (b) an oligonucleotide tail;    (d) amplifying the products of step (c) using 
 (i) a first primer which is complementary to said Type IIS restriction site-containing sequence; and  
 (ii) a second primer which is complementary to said oligonucleotide tail; and  
 (e) treating the products of step (d) with a Type IIS restriction enzyme that recognizes said Type IIS restriction site to cleave said products, thereby removing the 3′-untranslated regions and stop codons.  
   
     
     
         15 . The library of nucleic acid molecules of  claim 14 , produced by the further steps of: 
 (f) ligating a sequence which encodes an affinity tag to the cleaved ends of the products of step (e);    (g) transcribing the products of step (f);    (h) ligating peptidyl acceptors to the 3′ ends of the RNA products of step (g);    (i) translating said products of step (h) to produce a population of RNA-protein fusions; and    (j) substantially isolating RNA-protein fusions which comprise said affinity tag, thereby obtaining a population of mRNA molecules lacking 3′-untranslated regions and stop codons.    
     
     
         16 . A library of nucleic acid molecules produced by the steps of: 
 (a) providing a population of mRNA molecules;    (b) synthesizing strands of DNA, each of which is complementary to one of said mRNA molecules, using a random primer mixture, said random primer mixture comprising primers, each having (i) a 5′ region which lacks a stop codon in at least one reading frame and (ii) a random 3′ region; and    (c) synthesizing strands of DNA complementary to said DNA strands of step (b), using a second random primer mixture.