Method for generating aptamers with improved off-rates

• US 10,316,321 B2
• Filed: 01/23/2018
• Issued: 06/11/2019
• 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
• Application Number:
  US15/878,161
• Publication Number:
  US 20180155725A1
• Time in Patent Office: 504 Days
• Field of Search: None
• 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. A mixture comprising HSA, casein and prothrombin.

2. The mixture of claim 1, wherein the mixture is a buffer solution.

3. The mixture of claim 2, wherein the pH of the buffer solution is about 7.5.

4. The mixture of claim 1, wherein HSA is at about 0.1%.

5. The mixture of claim 1, wherein casein is at about 10 μ

M.

6. The mixture of claim 1, wherein prothrombin is at about 10 μ

M.

7. The mixture of claim 1 further comprising a nucleic acid molecule.

8. The mixture of claim 7, wherein the nucleic acid molecule is an aptamer.

9. A formulation comprising HEPES, NaCl, KCl, MgCl₂, EDTA and a polysorbate-20.

10. The formulation of claim 9, wherein HEPES is at about 40 mM.

11. The formulation of claim 9, wherein NaCl is at about 125 mM.

12. The formulation of claim 9, wherein KCl is at about 5 mM.

13. The formulation of claim 9, wherein MgCl₂ is at about 5 mM.

14. The formulation of claim 9, wherein EDTA is at about 1 mM.

15. The formulation of claim 9, wherein polysorbate 20 is at about 0.05%.

16. The formulation of claim 9, wherein the pH of the formulation is about 7.5.

17. The formulation of claim 9 further comprising a nucleic acid molecule.

18. The formulation of claim 17, wherein the nucleic acid molecule is an aptamer.