Systematic evolution of ligands by exponential enrichment: Chemi-SELEX

Abstract

This application provides methods for identifying nucleic acid ligands capable of covalently interacting with targets of interest. The nucleic acids can be associated with various functional units. The method also allows for the identification of nucleic acids that have facilitating activities as measured by their ability to facilitate formation of a covalent bond between the nucleic acid, including its associated functional unit, and its target.

1 . A method for preparing nucleic acid ligands that bind covalently with a target compound comprising: 
 a) identifying a nucleic acid ligand of a target compound from a candidate mixture of nucleic acids by a method comprising: i) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; ii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, and iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target may be identified; and    b) attaching at least one chemically reactive functional unit which covalently binds to a target to said nucleic acid ligand to yield a nucleic acid ligand of the target compound.    
     
     
         2 . The method of  claim 1  wherein said chemically reactive functional unit is selected from the group consisting of photoreactive groups, active site directed compounds and peptides.  
     
     
         3 . A nucleic acid ligand prepared according to the method of  claim 1 .  
     
     
         4 . The nucleic acid ligand of  claim 3  wherein said at least one functional unit is attached to an oligonucleotide capable of hybridizing to said nucleic acid ligand, and wherein step b) is accomplished by hybridizing said oligonucleotide to said nucleic acid ligand.  
     
     
         5 . A method for identifying a diagnostic reagent of a target from a candidate mixture comprised of nucleic acids each having at least one nucleic acid region and at least one chemically reactive functional unit, said method comprising: 
 a) contacting said candidate mixture with said target molecule, wherein nucleic acids which bind covalently with said target molecule form nucleic acid-target molecule complexes;    b) partitioning the nucleic acid-target molecule complexes from the remainder of the candidate mixture, whereby nucleic acid ligands that bind covalently with the target molecule are identified.    
     
     
         6 . The method of  claim 5  wherein after step b), the nucleic acid is amplified and steps a ) and b) are repeated until a mixture of nucleic acids enriched in sequences that bind covalently with the target molecule is obtained.  
     
     
         7 . The method of  claim 5  wherein said chemically reactive functional unit is selected from the group consisting of photoreactive groups, active site directed compounds and peptides.  
     
     
         8 . The method of  claim 5  wherein at least one nucleic acid region is comprised of a fixed region and a randomized region.  
     
     
         9 . The method of  claim 8  wherein at least one said chemically reactive functional unit is attached to an oligonucleotide hybridized to said fixed region.  
     
     
         10 . The method of  claim 5  further comprising adding a reporter molecule to the diagnostic reagent.  
     
     
         11 . A diagnostic reagent identified according to the method of  claim 5 .  
     
     
         12 . A method for preparing a diagnostic reagent, wherein said diagnostic reagent comprises a nucleic acid ligand that binds covalently with a target compound and a reporter molecule, comprising: 
 a) identifying a nucleic acid ligand of a target compound from a candidate mixture of nucleic acids by a method comprising: i) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; ii) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, and iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target may be identified; and    b) attaching at least one chemically reactive functional unit which covalently binds to a target to said nucleic acid ligand to yield a nucleic acid ligand of the target compound; and    c) attaching at least one reporter molecule to said nucleic acid ligand, whereby a diagnostic reagent is prepared.    
     
     
         13 . The method of  claim 12  wherein said chemically reactive functional unit is selected from the group consisting of photoreactive groups, active site directed compounds and peptides.  
     
     
         14 . A diagnostic reagent prepared by the method of  claim 12.