Selex and photoSELEX

• US 8,409,795 B2
• Filed: 07/17/2008
• Issued: 04/02/2013
• Est. Priority Date: 07/17/2007
• Status: Active Grant

• Current Assignee: SomaLogic Operating Co., Inc. (Somalogic Incorporated)
• Original Assignee: Somalogic Incorporated
• Inventors: Schneider, Daniel J., Wilcox, Sheri K., Zichi, Dominic, Nieuwlandt, Dan, Carter, Jeff, Gold, Larry
• Primary Examiner(s): Chong, Kimberly
• Application Number:
  US12/175,388
• Publication Number:
  US 20090098549A1
• Time in Patent Office: 1,720 Days
• Field of Search: None
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2541/101 Selex

Abstract

The present disclosure describes improved SELEX methods for generating nucleic acid ligands that are capable of binding to target molecules and improved photoSELEX methods for generating photoreactive nucleic acid ligands that are capable of both binding and covalently crosslinking to target molecules. The disclosure further describes nucleic acid libraries having expanded physical and chemical properties and their use in SELEX and photoSELEX; methods for increasing the crosslinking efficiencies of photoaptamers; methods for producing photoaptamers having selective modifications that enhance functionality and minimize non-specific photoreactions; and methods for generating truncated nucleic acid ligands from nucleic acid ligands of longer length. The disclosure further describes aptamers and photoaptamers obtained by using any of the foregoing.

Claims

1. A method for identifying a photoaptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids, wherein each nucleic acid comprises;

i) at least one non-photoreactive placeholding modified pyrimidine; and

ii) at least one modified pyrimidine independently selected from the group consisting of the modified pyrimidines having the following structure;

2. The method of claim 1 wherein said non-photoreactive placeholding modified pyrimidine comprises a methyl group at the C-5 position and said photoreactive pyrimidine comprises a Br or I at the C-5 position.

3. The method of claim 1 wherein said non-photoreactive placeholding modified pyrimidine is 5-methyl-cytosine.

4. The method of claim 1 wherein said non-photoreactive placeholding modified pyrimidine is thymine.

5. The method of claim 1 wherein said photoreactive pyrimidine is 5-Br-cytosine.

6. The method of claim 1 wherein said photoreactive pyrimidine is 5-Br-uracil.

7. A method for identifying a photoaptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids comprising at least one non-photoreactive placeholding modified pyrimidine;

b) contacting the candidate mixture with a target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

d) amplifying the increased affinity nucleic acids to yield a nucleic acid ligand-enriched mixture of nucleic acids;

e) repeating (b)-(d) as desired;

f) producing from said nucleic acid ligand-enriched mixture of nucleic acids a candidate photoaptamer or a mixture of candidate photoaptamers by replacing in each nucleic acid of the ligand-enriched mixture of nucleic acids one or more non-photoreactive placeholding pyrimidines with a photoreactive pyrimidine;

g) contacting said candidate photoaptamer(s) with said target wherein a candidate photoaptamer-target complex is formed;

h) irradiating said candidate photoaptamer-target complex;

i) determining whether said candidate photoaptamer-target complex has photocrosslinked;

j) repeating (f)-(i) as desired; and

k) identifying at least one photoaptamer to the target.

9. The method of claim 7 wherein said non-photoreactive placeholding modified pyrimidine comprises a methyl group at the C-5 position and said photoreactive pyrimidine comprises a Br or I at the C-5 position.

10. The method of claim 7 wherein said non-photoreactive placeholding modified pyrimidine is 5-methyl-cytosine.

11. The method of claim 7 wherein said non-photoreactive placeholding modified pyrimidine is thymine.

12. The method of claim 7 wherein said photoreactive pyrimidine is 5-Br-cytosine.

13. The method of claim 7 wherein said photoreactive pyrimidine is 5-Br-uracil.

8. A method for identifying an aptamer, the method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture with a biotinylated target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to other nucleic acids in the candidate mixture bind the target molecule, forming nucleic acid-target molecule complexes;

c) partitioning the nucleic acid-target molecule complexes from the candidate mixture by contacting the candidate mixture with a solid support having streptavidin bound to the solid support and washing the solid support with a solution containing biotin;

d) dissociating the nucleic acid-target molecule complexes to form free nucleic acids;

e) amplifying the free nucleic acids to yield a mixture of nucleic acids enriched with nucleic acids that are capable of binding to the target molecule with increased affinity;

f) repeating (b) through (e) as desired; and

g) identifying at least one aptamer to the target molecule.