Method of measuring the affinity of biomolecules

• US 8,778,697 B2
• Filed: 11/10/2006
• Issued: 07/15/2014
• Est. Priority Date: 11/16/2005
• Status: Expired due to Fees

• Current Assignee: Belluscura Limited
• Original Assignee: Nanotether Discovery Science Limited (Belluscura Plc)
• Inventors: Dale, Trevor Clive, Borri, Paola, Harwood, Adrian John
• Primary Examiner(s): YAKOVLEVA, GALINA M
• Application Number:
  US12/094,073
• Publication Number:
  US 20090023227A1
• Time in Patent Office: 2,804 Days
• Field of Search: None
• US Class Current: 436/501
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/557 using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction

Abstract

The invention provides a method of measuring the affinity of first and second biomolecules in which a first biomolecule is tethered by a first tether portion having a first tether portion length and a second biomolecule is tethered by a second tether portion having a second tether portion length, the method comprising determining binding of adjacent first and second biomolecules to each other, varying at least one of the first and second tether lengths and determining binding of the first and second biomolecules. The invention also provides apparatus suitable for use in the method of the invention.

Claims

1. A method of measuring the affinity of a first and a second biomolecule, wherein the first biomolecule is tethered by a first tether portion having a first tether portion length and a first tether persistence length and the second biomolecule is tethered by a second tether portion having a second tether portion length and a second tether persistence length, wherein the first and second biomolecules are tethered to a surface, such that swept volumes defined by the movement of each biomolecule overlap, so that the first and second biomolecules are able to bind to each other, said method comprising determining a proportion of the first and second biomolecules bound to each other.

2. The method according to claim 1, wherein a range of the first and second tether portion lengths produce a range of effective concentrations for the first and/or second biomolecules.

3. The method according to claim 2, wherein said method further comprises determining the binding of the first and second biomolecules in the presence of a third biomolecule.

4. The method according to claim 2, wherein the length of the first and/or second tether portion are different.

5. The method according to claim 1, wherein the at least one tether portion comprises nucleotides.

6. The method according to claim 1, wherein the at least one tether portion comprises a double-stranded DNA.

7. The method according to claim 1, wherein the at least one tether portion comprises a carbon nanotube, an amyloid fibril, or a polymer.

8. The method according to claim 7, wherein the polymer is a DNA crossover complex.

9. The method according to claim 1, wherein said first and/or second tether portions are made from double-stranded DNA, DX hybrids, carbon nanotubes, amyloid fibrils, or polymers to produce a range of effective concentrations for the first and/or second biomolecules.

10. The method according to claim 1, wherein the proportion of the first and second biomolecules that are molecularly close to each other indicates the proportion of interacting the first and second biomolecules.

11. The method according to claim 1, wherein the proportion of binding of the first and second biomolecules is determined by the intensity of fluorescence (Fö

rster) resonance energy transfer (FRET) between a first and second fluorophores respectively attached to, or integrated with, the first and second biomolecules.

12. The method according to claim 1, wherein the K_d of an interaction between the first and second biomolecules is determined by determining the proportion of the first and second biomolecules bound to each other for a range of concentrations of the first and second biomolecules and determining the concentration of the first or second biomolecule required for half maximal binding of the first and second biomolecules.

13. The method according to claim 1, wherein the K_off value for an interaction between the first and second biomolecules is determined by providing initial saturating concentrations of the first and second biomolecules, cleaving the second tether portion and monitoring any change in levels of bound first and second biomolecules.

14. The method according to claim 1, wherein said method further comprises providing a concentration of the first and second biomolecules around the K_d of an interaction between the first and second biomolecules and determining the effect of a modulator on the portion of the first and the second biomolecules bound to each other.

15. The method according to claim 1, wherein the first and second tether portion are linked together to form a Y-shaped tether.

16. The method according to claim 1, wherein the first and second biomolecules are tethered to the surface at an inter-anchor distance so that the first and second biomolecules are closely adjacent to each other.

17. The method according to claim 16, wherein a range of inter-anchor distances produce a range of effective concentrations for the first and/or second biomolecules.

18. The method according to claim 1, wherein the second biomolecule is selected from a library.

19. The method according to claim 1, wherein the method further comprises changing said first and/or second persistence length of said first and/or second tethers of the first and/or second tether portion by chemical modification or physical association of the first and/or second tether portion to produce a range of effective concentrations for the first and/or second biomolecules.

20. The method according to claim 1, wherein the surface is provided by a solid support.

21. The method according to claim 20, wherein the at least one tether portion is tethered to the solid support by an oligonucleotide anchor.

22. The method according to claim 21, wherein the oligonucleotide anchor is set up as an array on the solid support.

23. The method according to claim 20, wherein the solid support is a glass slide.

24. The method according to claim 23, wherein the tether is set up as an array on the solid support.

25. The method according to claim 1, wherein the surface is provided by a microbead.