Method for generating aptamers with improved off-rates

• US 8,404,830 B2
• Filed: 12/15/2010
• Issued: 03/26/2013
• Est. Priority Date: 07/17/2007
• Status: Active Grant

• Current Assignee: SomaLogic Operating Co., Inc. (Somalogic Incorporated)
• Original Assignee: Somalogic Incorporated
• Inventors: Zichi, Dominic, Wilcox, Sheri K., Bock, Chris, Schneider, Daniel J., Eaton, Bruce, Gold, Larry
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US12/969,239
• Publication Number:
  US 20110082286A1
• Time in Patent Office: 832 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/205 Aptamer

Abstract

The present disclosure is directed to the field of nucleic acid chemistry, specifically to 5-position modified uridines, as well as, oligonucleotides comprising said 5-position modified uridines. The present disclosure describes methods for producing aptamers comprising said 5-position modified uridines that are capable of binding to target molecules.

Claims

1. A compound having the following structure:

2. An oligonucleotide comprising at least one modified nucleotide having the following structure:

3. The oligonucleotide of claim 2, wherein the oligonucleotide is a mixed ribonucleic acid/deoxyribonucleic acid or a deoxyribonucleic acid.

4. The oligonucleotide of claim 3, wherein said oligonucleotide 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-azido-9-hypoxanthinyl, 8-bromo-9-hypoxanthinyl, 8-iodo-9-hypoxanthine, 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-(8-azido-9-hypoxanthinyl)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.

5. The oligonucleotide of claim 3, wherein said carbohydrate moiety has 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′

—

NH₂) and 2′

-azido.

6. The oligonucleotide of claim 3, wherein said carbohydrate moiety has been replaced by an epimeric sugar moiety selected from the group consisting of 1-arabinosyl, 1-xylosyl and 1-lyxosyl and α

-anomeric analogs thereof.

7. The oligonucleotide of claim 3, wherein said oligonucleotide has at least one additional modification which is modification which is a backbone modification of one or more phosphate moieties selected from the group consisting of P(O)S (“

thioate”

), P(S)S (“

dithioate”

), P(O)(NR₂) (“

amidate”

), P(O)R, P(O)OR′

, CO and CH₂, wherein R and R′

are independently H, aryl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl and C_1-20 alkyl, optionally containing an ether (—

O—

) linkage.

8. An aptamer comprising at least one modified nucleotide having the following structure:

9. The aptamer of claim 8, wherein the aptamer is a mixed ribonucleic acid/deoxyribonucleic acid or a deoxyribonucleic acid.

10. The oligonucleotide of claim 9, wherein said oligonucleotide 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-azido-9-hypoxanthinyl, 8-bromo-9-hypoxanthinyl, 8-hypoxanthinyl, 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-indol-3-yl)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.

11. The oligonucleotide of claim 9, wherein said carbohydrate moiety has 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′

—

NH₂) and 2′

-azido.

12. The oligonucleotide of claim 9, wherein said carbohydrate moiety has been replaced by an epimeric sugar moiety selected from the group consisting of 1-arabinosyl, 1-xylosyl and 1-lyxosyl and α

-anomeric analogs thereof.

13. The oligonucleotide of claim 9, wherein said oligonucleotide has at least one additional modification which is a backbone modification of one or more phosphate moieties selected from the group consisting of P(O)S (“

thioate”

), P(S)S (“

dithioate”

), P(O)(NR₂) (“

amidate”

), P(O)R, P(O)OR′

, CO and CH₂, wherein R and R′

are independently H, aryl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl and C_1-20 alkyl, optionally containing an ether (—

O—

) linkage.