Truncation SELEX method

Abstract

This invention is directed to a method for identifying nucleic acid ligands by the SELEX method wherein the participation of fixed sequences is eliminated or minimized.

1 - 10 . (canceled)  
     
     
         11 . A method for identifying nucleic acid ligands of a target compound, said method comprising: 
 a) providing an candidate mixture of nucleic acids of differing sequences comprising randomized sequences,    b) contacting said candidate mixture with a target under conditions favorable for binding between the target and members of the candidate mixture;    c) partitioning those nucleic acids with higher affinity for the target from those nucleic acids with lesser affinity to the target to generate an enriched candidate mixture;    d) ligating nucleic acids in said enriched candidate mixture to a 3′ primer binding site using RNA ligase to generate a ligation product;    e) reverse transcribing the ligation product using the 3′ primer to generate cDNA;    f) ligating the cDNA to a DNA oligonucleotide encoding the T7 promoter to generate a new dsDNA candidate mixture;    g) transcribing the new candidate mixture to generate a new RNA candidate mixture;    h) removing the 3′ primer binding site to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target compound may be identified.    
     
     
         12 . The method of  claim 11  further comprising the step: 
 i) repeating steps b) through h) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of ligand enrichment, whereby nucleic acid ligands of the target compound may be identified.    
     
     
         13 . The method of  claim 11  wherein said candidate mixture is prepared from a template comprising a region of conserved sequence and a region of randomized sequence.  
     
     
         14 . The method of  claim 13 , wherein said conserved sequence is a restriction site, and wherein said preparation comprises digesting the candidate mixture with a restriction enzyme and transcribing the digested candidate mixture.  
     
     
         15 . The method of  claim 11  wherein said candidate mixture is prepared by providing a candidate mixture of nucleic acids of differing sequences comprising 3′-region of conserved sequence, 5′-region of conserved sequence, and a region of randomized sequence, and removing the 3′-region of conserved sequence and the 5′-region of conserved sequence by enzymatic digestion.  
     
     
         16 . The method of  claim 15 , comprising: 
 a) providing an RNA candidate mixture comprising a 3′-region of conserved sequence, a 5′-region of conserved sequence and a region of randomized sequence;    b) generating a cDNA candidate mixture from the RNA candidate mixture;    c) transcribing the cDNA candidate mixture to generate an RNA transcript;    d) removing the 5′ region of conserved sequence with RNaseH to generate digested RNA;    e) reverse transcribing the digested RNA to generate cDNA;    f) optionally ligating a 3′-primer binding site to the cDNA;    g) ligating the generated cDNA to a DNA oligonucleotide encoding the T7 promoter-initiator to generate a ligation product;    h) amplifying the ligation product to generate a new dsDNA candidate mixture lacking at least part of the 5′-region of conserved sequence;    i) transcribing the dsDNA to generate a 5′-truncated transcript; and    j) digesting the 3′end of the 5′-truncated transcript to generate a truncated RNA candidate mixture.    
     
     
         17 . The method of  claim 16 , wherein the 3′-region of conserved sequence comprises a primer binding site.  
     
     
         18 . The method of  claim 17 , wherein step d) further comprises removing the 3′ region of conserved sequence with RNaseH.