Method for Generating Aptamers with Improved Off-Rates

Abstract

The present disclosure describes improved SELEX methods for producing aptamers that are capable of binding to target molecules and improved photoSELEX methods for producing photoreactive aptamers that are capable of both binding and covalently crosslinking to target molecules. Specifically, the present disclosure describes methods for producing aptamers and photoaptamers having slower dissociation rate constants than are obtained using prior SELEX and photoSELEX methods. The disclosure further describes aptamers and photoaptamers having slower dissociation rate constants than those obtained using prior methods. In addition, the disclosure describes aptamer constructs that include a variety of functionalities, including a cleavable element, a detection element, and a capture or immobilization element.

What is claimed is: 
     
         1 . An aptamer that binds specifically to a target, wherein the aptamer comprises at least one base-modified nucleotide, the base-modified nucleotide having the following structure: 
       
         
           
           
               
               
           
         
       
       and wherein Z═R plus (CH 2 ) n  connecting group, where n=1, 2 or 3, and further wherein R is: 
       
         
           
           
               
               
           
         
       
     
     
         2 . The aptamer of  claim 1  wherein n=1 or 2. 
     
     
         3 . The aptamer of  claim 1  wherein the aptamer comprises at least two of said base-modified nucleotides. 
     
     
         4 . The aptamer of  claim 1  wherein the aptamer comprises at least three of said base-modified nucleotides. 
     
     
         5 . The aptamer of  claim 1  wherein the aptamer has a rate of dissociation (t 1/2 ) from a non-covalent aptamer-target complex of greater than or equal to 30 minutes. 
     
     
         6 . A non-covalent complex of the aptamer of  claim 1  and a target molecule. 
     
     
         7 . A non-covalent complex according to  claim 6 , wherein the rate of dissociation (t 1/2 ) of the aptamer from the target molecule is greater than or equal to 30 minutes. 
     
     
         8 . A non-covalent complex according to  claim 6  wherein the target molecule is a protein or peptide. 
     
     
         9 . A non-covalent complex according to  claim 8  wherein the target molecule is a protein selected from  FIG. 7 . 
     
     
         10 . The aptamer of  claim 1 , wherein the aptamer comprises a ribonucleic acid, a ribonucleic acid and a deoxyribonucleic acid, or a deoxyribonucleic acid. 
     
     
         11 . The aptamer of  claim 1 , wherein said aptamer has at least one additional modified base selected from the group consisting of 5-bromo-1-uracilyl, 5-iodo-1-uracilyl, 5-bromovinyl-1-uracilyl, 5-iodovinyl-1-uracilyl, 5-azido-1-uracilyl, 4-thio-1-uracilyl, 4-thio-1-cytosinyl, 5-bromo-1-cytosinyl, 5-iodo-1-cytosinyl, 5-bromovinyl-1-cytosinyl, 5-iodovinyl-1-cytosinyl, 5-azido-1-cytosinyl, 8-azido-9-adeninyl, 8-bromo-9-adeninyl, 8-iodo-9-adeninyl, 8-azido-9-guaninyl, 8-bromo-9-guaninyl, 8-iodo-9-guaninyl, 8-azidohypoxanthinyl, 8-bromohypoxanthinyl, 8-iodohypoxanthinyl, 8-azido-9-xanthinyl, 8-bromo-9-xanthinyl, 8-iodo-9-xanthinyl, 5-[(4-azidophenacyl)thio]-1-cytosinyl, 5-[(4-azidophenacyl)thio]-1-uracilyl, 5-N-(benzylcarboxamido)-1-uracilyl, 5-(N-isobutylcarboxamido)]-1-uracilyl, 5-(N-tryptaminocarboxyamido)-1-uracilyl, 5-(N-[2-(1H-indole-3yl)ethyl]carboxamido)-1-uracilyl, 5-(N-[1-(3-trimethylammonium)propyl]carboxamido)-1-uracilyl chloride, 5-(N-naphthylmethylcarboxamido)-1-uracilyl, 5-(N-[1-(2,3-dihydroxypropyl)]carboxamido)-1-uracilyl, 7-deaza-7-iodo-9-adeninyl, 7-deaza-7-iodo-9-guaninyl, 7-deaza-7-bromo-9-adeninyl, 7-deaza-7-bromo-9-guaninyl, 1-isocytidinyl and 9-isoguaninyl. 
     
     
         12 . The aptamer of  claim 1 , wherein said aptamer further comprises a sugar modification selected from the group consisting of 2′-methyl, 2′-O-methyl (2′-OMe), 2′-O-allyl, 2′-fluoro (2′-F), 2′-amino (2′-NH 2 ) and 2′-azido. 
     
     
         13 . The aptamer of  claim 1 , wherein said aptamer comprises at least one additional modification comprising a backbone modification of the phosphate moiety selected from the group consisting of P(O)S (“thioate”), P(S)S (“dithioate”), P(O)(NR 2 ) (“amidate”), P(O)R, P(O)OR′, CO and CH 2 , wherein R and R′ are independently H, aryl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl and C 1-20  alkyl, optionally containing an ether (—O—) linkage. 
     
     
         14 . A compound having the following structure: 
       
         
           
           
               
               
           
         
       
       and wherein Z═R plus (CH 2 ) n  connecting group, where n=1, 2 or 3, and further wherein R is: 
       
         
           
           
               
               
           
         
       
     
     
         15 . The compound of  claim 14  further comprising a sugar modification selected from the group consisting of 2′-methyl, 2′-O-methyl (2′-OMe), 2′-O-allyl, 2′-fluoro (2′-F), 2′-amino (2′-NH 2 ) and 2′-azido. 
     
     
         16 . The compound of  claim 14  further comprising a backbone modification of the phosphate moiety selected from the group consisting of P(O)S (“thioate”), P(S)S (“dithioate”), P(O)(NR 2 ) (“amidate”), P(O)R, P(O)OR′, CO and CH 2 , wherein R and R′ are independently H, aryl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl and C 1-20  alkyl, optionally containing an ether (—O—) linkage. 
     
     
         17 . A method for identifying an aptamer capable of binding to a target molecule, the method comprising:
 (a) contacting a candidate mixture wherein the candidate mixture comprises modified nucleic acids in which one, several or all pyrimidines in at least one, or each, nucleic acid is chemically modified at the 5-position wherein the modified pyrimidine is selected from the group consisting of 5-[N-(benzyl)carboxamide]-2′-deoxyuridine, 5-[N-(phenyl-2-ethyl)carboxamide]-2′-deoxyuridine, and 5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxyuridine, with the target molecule to form a sample mixture, and allowing for the formation of a nucleic acid-target complex, wherein a nucleic acid-target complex forms when a nucleic acid has affinity for the target;   (b) diluting the sample mixture;   (c) partitioning the nucleic acid-target molecule complex from the remainder candidate mixture;   (d) dissociating the nucleic acid-target molecule complex to generate free nucleic acid, and wherein at least one nucleic acid from the free nucleic acid is capable of binding to the target molecule,   whereby an aptamer to the target molecule may be identified.   
     
     
         18 . The method of  claim 17 , further comprising:
 a. (i) diluting the candidate mixture containing the nucleic acid-target molecule complex; and (ii) incubating the diluted candidate mixture containing the nucleic acid-target molecule complex;   b. (i) mixing the candidate mixture containing the nucleic acid-target molecule complex with at least one competitor molecule wherein the competitor molecule comprises a polyanion; and (ii) incubating the candidate mixture containing the competitor and the nucleic acid-target molecule complex; or   c. (i) mixing the candidate mixture containing the nucleic acid-target molecule complex with at least one competitor molecule wherein the competitor molecule comprises a polyanion; (ii) before, simultaneous with, or after introduction of the competitor molecule, diluting the candidate mixture containing the nucleic acid-target molecule complex; and (iii) incubating the diluted candidate mixture containing the competitor and the nucleic acid-target molecule complex.   
     
     
         19 . An aptamer comprising a sequence of an aptamer identified by the method of  claim 17 .