Patent ID: 8101385

Claim:
A method of identifying an aptamer, wherein the aptamer comprises a 2′-OMe modified nucleotide, comprising: a) preparing a transcription reaction mixture comprising (i) a modified T7 RNA polymerase that is able to incorporate any one of a 2′-OMe modified nucleotide triphosphate (2′-OMe NTP) selected from 2′-OMe ATP, 2′-OMe GTP, 2′-OMe CTP, 2′-OMe TTP and 2′-OMe UTP, wherein the modified T7 RNA polymerase exhibits an increased ability to incorporate a 2′-modified nucleotide triphosphate (2′-modified NTP) as compared to the ability of the corresponding unmodified T7 RNA polymerase to incorporate the 2′-modified NTP, wherein the modified T7 RNA polymerase comprises mutations at amino acid positions 639 and 784 relative to the wild-type T7 RNA polymerase of SEQ ID NO: 33 and wherein the amino acid at position 639 is changed to a leucine and the amino acid at position 784 is changed to an alanine; (ii) at least one nucleic acid transcription template; and (iii) nucleotide triphosphates (NTPs), wherein the nucleotide triphosphates comprise guanidine triphosphates and wherein all of the guanidine triphosphates are 2′-OMe modified, and wherein the mixture of nucleoside triphosphates is selected from the following: a mixture of 2′-deoxy cytidine triphosphate (2′-deoxy CTP), 2′-OMe adenosine triphosphate (2′-OMe ATP), 2′-OMe guanosine triphosphate GTP (2′-OMe GTP), and 2′-OMe uridine triphosphate (2′-OMe UTP) or 2′-OMe thymidine nucleotide triphosphates (2′-OMe TTP); a mixture of 2′-deoxy thymidine triphosphate (2′-deoxy TTP) or 2′-deoxy uridine triphosphate (2′-deoxy UTP), and 2′-OMe adenosine triphosphate (2′-OMe ATP), 2′-OMe guanosine triphosphate GTP (2′-OMe GTP), and 2′-OMe cytidine triphosphate (2′-OMe CTP), and a mixture of 2′-OH thymidine triphosphate (2′-OH TTP) or 2′-OH uridine triphosphate (2′-OH UTP), and 2′-OMe adenosine triphosphate (2′-OMe ATP), 2′-OMe guanosine triphosphate GTP 2′-OMe GTP), and 2′-OMe cytidine triphosphate (2′-OMe CTP); b) preparing a candidate mixture of single-stranded nucleic acids by transcribing the transcription reaction mixture under conditions whereby the modified T7 RNA polymerase incorporates at least one 2′-OMe modified nucleotide triphosphate into the nucleic acids of the candidate mixture of single-stranded nucleic acids, wherein all guanidine nucleotides of the single-stranded nucleic acids of the candidate mixture after the initial guanosine nucleotide are 2′-OMe modified; c) contacting the candidate mixture with a target molecule; d) partitioning the nucleic acids having an increased affinity for the target molecule from the candidate mixture, relative to the affinity of the candidate mixture for the target molecule; and e) amplifying the increased affinity nucleic acids to yield an aptamer enriched mixture, whereby an aptamer to the target molecule comprising all 2′-OMe-modified guanidine nucleotides, except optionally one of the guanidine nucleotides of the aptamer, is identified.