Method for generating aptamers with improved off-rates

• US 8,975,388 B2
• Filed: 05/23/2011
• Issued: 03/10/2015
• Est. Priority Date: 01/16/2007
• 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): VIVLEMORE, TRACY ANN
• Application Number:
  US13/113,261
• Publication Number:
  US 20110245479A1
• Time in Patent Office: 1,387 Days
• Field of Search: None
• US Class Current: 536/23.1
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/334 Modified C
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: C12N 9/6429 Thrombin (3.4.21.5)
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2537/101 : Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2565/514 : characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 33/5308 : for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence

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.

Claims

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:

2. The aptamer of claim 1, wherein the aptamer has a slow rate of dissociation from its target wherein said rate of dissociation (t_1/2) is greater than or equal to about 30 minutes.

3. The aptamer of claim 2 wherein said rate of dissociation (t_1/2) is between about 30 minutes and about 240 minutes.

4. The aptamer of claim 2 wherein said rate of dissociation (t_1/2) is selected from the group consisting of a time ≧

about 30 minutes, ≧

about 60 minutes, ≧

about 90 minutes, ≧

about 120 minutes, ≧

about 150 minutes, ≧

about 180 minutes, ≧

about 210 minutes and ≧

about 240 minutes.

5. The aptamer of claim 2 wherein the target is selected from the group consisting of the targets listed in FIG. 7.

6. The aptamer of claim 2 wherein said target is a protein.

7. The aptamer of claim 2 wherein said target is a peptide.

8. The aptamer of claim 2 wherein said aptamer comprises at least one element independently selected from the group consisting of:

i) a cleavable element,

ii) a detectable element,

iii) a spacer element, and

iv) a tag.

9. A biochip comprising an aptamer of claim 1.

10. A diagnostic device comprising an aptamer of claim 1.

11. A biosensor comprising an aptamer of claim 1.

12. An ALONA device comprising an aptamer of claim 1.

13. The aptamer of claim 1 wherein n=1.

14. The aptamer of claim 1 wherein n=2.

15. The aptamer of claim 1 wherein n=3.

16. The aptamer of claim 1 wherein the aptamer comprises at least two of said base-modified nucleotides.

17. The aptamer of claim 1 wherein the aptamer comprises at least three of said base-modified nucleotides.

18. The aptamer of claim 1, wherein the aptamer is a mixed ribonucleic acid/deoxyribonucleic acid or a deoxyribonucleic acid.

19. 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-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.

20. The aptamer of claim 1, wherein said aptamer further comprises at least one additional chemical modification.

21. The aptamer of claim 20, wherein said at least one additional chemical modification is a chemical substitution at one or more positions independently selected from the group consisting of a ribose position, a deoxyribose position, a phosphate position, and a base position.

22. The aptamer of claim 20, wherein said at least one additional chemical modification is independently selected from the group consisting of a 2′

-methyl, a 2′

-amino (2′

-NH₂), a 2′

-fluoro (2′

-F), a 2′

-O-methyl (2′

-OMe), a 2′

-O-allyl, a 2′

-azido, a modification at a cytosine exocyclic amine, a substitution of 5-bromouracil, a substitution of 5-bromodeoxyuridine, a substitution of 5-bromodeoxycytidine, a backbone modification, a methylation, a 3′

cap, and a 5′

cap.

23. The aptamer of claim 1, wherein R is 5-(N-naphthylmethylcarboxyamide)-2′

-deoxyuridine, 5-(N-naphthylethylcarboxyamide)-2′

-deoxyuridine, 5-(N-(2-naphthylmethyl)carboxyamide)-2′

-deoxyuridine or 5-(N-(2-naphthylethyl)carboxyamide)-2′

-deoxyuridine.

24. The aptamer of claim 1, wherein said aptamer further comprises at least one epimeric sugar moiety selected from the group consisting of 1-arabinosyl, 1-xylosyl, 1-lyxosyl and a-anomeric analogs thereof.

25. The aptamer of claim 1, wherein said aptamer has 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)(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.