Methods for identifying nucleic acid ligands

• US 8,236,570 B2
• Filed: 11/03/2009
• Issued: 08/07/2012
• Est. Priority Date: 11/03/2009
• Status: Active Grant

• Current Assignee: Vivonics Inc.
• Original Assignee: Infoscitex
• Inventors: Gilman, Vladimir Leo
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Pande, Suchira
• Application Number:
  US12/611,436
• Publication Number:
  US 20110104667A1
• Time in Patent Office: 1,008 Days
• Field of Search: None
• US Class Current: 436/161
• 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 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function

Abstract

The present invention generally relates to methods for identifying nucleic acid ligands of a target molecule. In certain embodiments, the invention provides methods for identifying a nucleic acid ligand of a target molecule from a candidate mixture of nucleic acids, including contacting at least one target molecule with a candidate mixture of nucleic acids, in which the nucleic acids have different affinities for the target molecule, and separating in a single step nucleic acids that bind the target molecule with greatest affinity from nucleic acids that bind the target molecule with a lesser affinity and nucleic acids that do not bind the target molecule, thereby identifying the nucleic acid ligand of the target molecule.

Claims

1. A method for identifying a nucleic acid ligand of a target molecule from a candidate mixture of nucleic acids, the method comprising:

incubating a candidate mixture of nucleic acids with a plurality of target molecules to form nucleic acid/target molecule complexes, wherein the target molecules are bound to beads; and

eluting the nucleic acids from the complexes that have been loaded onto an HPLC column by applying an HPLC gradient profile, wherein nucleic acids with the greatest affinity for the target molecule elute at an end portion of the gradient profile and nucleic acids with a lesser affinity for the target molecule and nucleic acids with no affinity for the target molecule elute prior to the end portion of the gradient profile, and wherein the gradient profile comprises a linearly increasing concentration of the target molecule and the end portion of the gradient profile comprises a linearly increasing concentration of the target molecule and a linearly increasing concentration of a chaotropic agent.

2. The method according to claim 1, wherein prior to incubating, the method further comprises loading the target molecules bound to the beads into an HPLC column.

3. The method according to claim 1, wherein subsequent to incubating, the method further comprises loading the candidate mixture and the nucleic acid/target molecule complexes onto an HPLC column.

4. The method according to claim 1, further comprising sequencing the nucleic acid ligand.

5. The method according to claim 4, wherein sequencing is a single-molecule sequencing by synthesis technique.

6. The method according to claim 1, wherein the target molecule is selected from the group consisting of:

a protein or portion thereof, an enzyme, a peptide, an enzyme inhibitor, a hormone, a carbohydrate, a glycoprotein, a lipid, a phospholipid, and a nucleic acid.

7. The method according to claim 6, wherein the protein is an infectious prion.

8. The method according to claim 1, wherein the nucleic acid ligand comprises DNA or RNA.