METHODS FOR IDENTIFYING NUCLEIC ACID LIGANDS

• US 20110104667A1
• Filed: 11/03/2009
• Published: 05/05/2011
• Est. Priority Date: 11/03/2009
• Status: Active Grant

• Current Assignee: Vivonics Inc.
• Original Assignee: Infoscitex
• Inventors: Gilman, Vladimir Leo
• Granted Patent: US 8,236,570 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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:

contacting at least one target molecule with a candidate mixture of nucleic acids, wherein 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 to the target molecule.

2. The method according to claim 1, wherein separating comprises:

loading the target molecule into a gradient gel;

applying an electric current to cause the target molecule to migrate to a position in the gel, wherein the target molecule remains immobilized at that position in the gel;

loading the candidate mixture into the gel; and

applying an electric current to cause the candidate mixture to migrate through the gel, wherein the nucleic acids with the greatest affinity for the target molecule bind to the target molecule immobilized in the gel, and the nucleic acids with lesser affinity for the target molecule and nucleic acids with no affinity for the target molecule migrate to an end of the gel.

3. The method according to claim 1, wherein separating comprises:

incubating the 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.

4. The method according to claim 3, wherein the gradient profile comprise 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.

5. The method according to claim 3, wherein prior to incubating, the method further comprises loading the target molecules bound to the beads into an HPLC column.

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

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

8. The method according to claim 7, wherein sequencing is a single-molecule sequencing by synthesis technique.

9. 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.

10. The method according to claim 9, wherein the protein is an infectious prion.

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

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

contacting a candidate mixture of nucleic acids to a plurality of target molecules under conditions to form a plurality of target/nucleic acid complexes, wherein the nucleic acids have different affinities for the target molecule and the nucleic acids that form the complex are the nucleic acids that have an increased affinity for the target molecule compared to a remainder of the nucleic acids in the mixture;

separating the target/nucleic acid complexes from the remainder of the mixture; and

dissociating the complexes in a manner in which bound nucleic acids dissociate from the target molecules at different rates based upon the different affinities of the bound nucleic acids to the target molecules, wherein nucleic acids that dissociate from the target molecule at the slowest rate are identified as the nucleic acid ligands of the target molecule.

13. The method according to claim 12, further comprising collecting the nucleic acid ligand.

14. The method according to claim 12, further comprising sequencing the nucleic acid ligand.

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

loading at least one target molecule into a gradient gel;

applying an electric current to cause the target molecule to migrate to a position in the gel, wherein the target molecule remains immobilized at that position in the gel;

loading a candidate mixture of nucleic acids into the gel; and

applying an electric current to cause the candidate mixture to migrate through the gel, wherein the nucleic acids with the greatest affinity for the target molecule bind to the target molecule immobilized in the gel, and the nucleic acids with lesser affinity for the target molecule and nucleic acids with no affinity for the target molecule migrate to an end of the gel.

16. The method according to claim 15, further comprising dissociating the bound nucleic acids from the target molecules.

17. The method according to claim 15, further comprising sequencing the nucleic acid ligand.

18. 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.

19. The method according to claim 18, wherein the gradient profile comprise 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.

20. The method according to claim 18, wherein prior to incubating, the method further comprises loading the target molecules bound to the beads into an HPLC column.

21. The method according to claim 18, wherein subsequent to incubating, the method further comprises loading the candidate mixture and the nucleic acid/target molecule complexes onto an HPLC column.

22. The method according to claim 18, further comprising sequencing the nucleic acid ligand.