Method for in vitro selection of 2'-substituted nucleic acids
First Claim
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1. A method for identifying nucleic acid ligands comprising a modified nucleotide to a target molecule comprising:
- a) preparing a transcription reaction mixture comprising a mutated polymerase, one or more 2′
-modified nucleotide triphosphates (NTPs), magnesium ions and one or more oligonucleotide transcription templates;
b) preparing a candidate mixture of single-stranded nucleic acids by transcribing the one or more oligonucleotide transcription templates under conditions whereby the mutated polymerase incorporates at least one of the one or more modified nucleotides into each nucleic acid of said candidate mixture, wherein each nucleic acid of said candidate mixture comprises a 2′
-modified nucleotide selected from the group consisting of a 2′
-position modified pyrimidine and a 2′
-position modified purine;
c) contacting the candidate mixture with said target molecule;
d) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture; and
e) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.
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Abstract
Materials and methods are provided for producing aptamer therapeutics having modified nucleotide triphosphates incorporated into their sequence. The aptamers produced by the methods of the invention have increased stability and half life.
91 Citations
76 Claims
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1. A method for identifying nucleic acid ligands comprising a modified nucleotide to a target molecule comprising:
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a) preparing a transcription reaction mixture comprising a mutated polymerase, one or more 2′
-modified nucleotide triphosphates (NTPs), magnesium ions and one or more oligonucleotide transcription templates;
b) preparing a candidate mixture of single-stranded nucleic acids by transcribing the one or more oligonucleotide transcription templates under conditions whereby the mutated polymerase incorporates at least one of the one or more modified nucleotides into each nucleic acid of said candidate mixture, wherein each nucleic acid of said candidate mixture comprises a 2′
-modified nucleotide selected from the group consisting of a 2′
-position modified pyrimidine and a 2′
-position modified purine;
c) contacting the candidate mixture with said target molecule;
d) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture; and
e) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method of preparing a nucleic acid comprising one or more modified nucleotides comprising:
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(a) preparing a transcription reaction mixture comprising a mutated polymerase, one or more 2′
-modified nucleotide triphosphates (NTPs), magnesium ions and one or more oligonucleotide transcription templates; and
(b) contacting the one or more oligonucleotide transcription templates with the mutated polymerase under conditions whereby the mutated polymerase incorporates the one or more 2′
-modified nucleotides into a nucleic acid transcription product. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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53. An aptamer composition comprising a sequence where substantially all adenosine nucleotides are 2′
- -OH adenosine, substantially all guanosine nucleotides are 2′
-OH guanosine, substantially all cytidine nucleotides are 2′
-O-methyl cytidine, and substantially all uridine nucleotides are 2′
-O-methyl uridine. - View Dependent Claims (54, 55, 56)
- -OH adenosine, substantially all guanosine nucleotides are 2′
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57. An aptamer composition comprising a sequence where substantially all purine nucleotides are 2′
- -deoxy purines and substantially all pyrimidine nucleotides are 2′
-O-methyl pyrimidines. - View Dependent Claims (58, 59, 60)
- -deoxy purines and substantially all pyrimidine nucleotides are 2′
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61. An aptamer composition comprising a sequence composition where substantially all guanosine nucleotides are 2′
- -OH guanosine, substantially all cytidine nucleotides are 2′
-O-methyl cytidine, substantially all uridine nucleotides are 2′
-O-methyl uridine, and substantially all adenosine nucleotides are 2′
-O-methyl adenosine. - View Dependent Claims (62, 63, 64)
- -OH guanosine, substantially all cytidine nucleotides are 2′
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65. An aptamer composition comprising a sequence where substantially all adenosine nucleotides are 2′
- -O-methyl adenosine, substantially all cytidine nucleotides are 2′
-O-methyl cytidine, substantially all guanosine nucleotides are 2′
-O-methyl guanosine or deoxy guanosine, substantially all uridine nucleotides are 2′
-O-methyl uridine, wherein less than about 10% of the guanosine nucleotides are deoxy guanosine. - View Dependent Claims (66, 67, 68)
- -O-methyl adenosine, substantially all cytidine nucleotides are 2′
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69. An aptamer composition comprising a sequence where substantially all adenosine nucleotides are 2′
- -O-methyl adenosine, substantially all uridine nucleotides are 2′
-O-methyl uridine, substantially all cytidine nucleotides are 2′
-O-methyl cytidine, and substantially all guanosine nucleotides are 2′
-F guanosine sequence. - View Dependent Claims (70, 71, 72)
- -O-methyl adenosine, substantially all uridine nucleotides are 2′
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73. An aptamer composition comprising a sequence where substantially all adenosine nucleotides are 2′
- -deoxy adenosine, substantially all cytidine nucleotides are 2′
-O-methyl cytidine, substantially all guanosine nucleotides are 2′
-O-methyl guanosine, and substantially all uridine nucleotides are 2′
-O-methyl uridine. - View Dependent Claims (74, 75, 76)
- -deoxy adenosine, substantially all cytidine nucleotides are 2′
Specification