METHOD

• US 20090023227A1
• Filed: 11/10/2006
• Published: 01/22/2009
• Est. Priority Date: 11/16/2005
• Status: Active Grant

• Current Assignee: Belluscura Limited
• Original Assignee: Nanotether Discovery Science Limited (Belluscura Plc)
• Inventors: Dale, Trevor Clive, Borri, Paola, Hardwood, Adrian John
• Granted Patent: US 8,778,697 B2
• Time in Patent Office: Days
• 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 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, determining binding of adjacent first and second biomolecules to each other, varying at least one of the first and second tether portion lengths and determining binding of the first and second biomolecules.

2. The method according to claim 1 in which the first and/or second biomolecules are tethered to a surface.

3. The method according to claim 2 in which both the first and the second biomolecules are tethered to a surface.

4. The method according to claim 3 in which the first and second biomolecules are tethered together and to the surface.

5. The method according to claim 2, in which the surface is provided by a solid support.

9. The method according to claim 1 in which the first and second tether portions are provided by a single tether.

10. The method according to claim 1 in which the first and second biomolecules are closely adjacent to each other, such that substantially hemispherical swept volumes defined by the movement of each biomolecule overlap, so that first and second biomolecules may bind to each other.

11. The method according to claim 1 in which by varying the first and second tether portion lengths the effective concentrations of the first and/or second biomolecules can be varied.

12. The method according to claim 11 in which a range of binding reactions that a third biomolecule affects is determined by determining binding of the first and second biomolecules.

19. The method according to claim 1 wherein at least one tether portion comprises nucleotides.

20. The method according to claim 19 in which the or each tether portion comprises double-stranded DNA.

24. The method according to claim 1 in which the, or each, tether comprises a carbon nanotube, amyloid fibril or a polymer.

25. The method according to claim 24 in which the polymer is a DNA crossover complex such as a DX hybrid.

31. The method according to any claim 2 in which at least one tether portion is tethered to the solid support by an oligonucleotide anchor.

34. The method according to claim 31 in which anchor oligonucleotides for the first and second tethers are synthesized in series.

42. The method according to claim 1 in which the proportion of the first and second biomolecules that are molecularly close to each other indicates the proportion of interacting first and second biomolecules.

43. The method according to claim 1 in which the proportion of binding first and second biomolecules is determined by the intensity of Forster resonance energy transfer (FRET) between first and second fluorophores respectively attached to, or integrated with, the first and second biomolecules.

45. The method according to claim 5 in which the solid support is a glass slide and oligonucleotide anchors or tethers are printed in arrays on a surface of the glass slide.

47. The method according to claim 10 in which the surface is provided by a microbead coupled to a specific first and second tethered biomolecule combination.

49. The method according to claim 19 in which at least one of the first and second biomolecules is a protein or polypeptide biomolecules connected to a first nucleic acid tether by an in vitro translation reaction to covalently attach a nascent peptide by its C-terminus close to, or at, the 3′

end of an mRNA-DNA conjugate.

57. The method according to claim 1 in which the K_d of an interaction between first and second biomolecules is determined by determining the proportion of bound first and second biomolecules for a range of concentrations of the first and second biomolecules and the determining the concentration (K_d) of the first or second biomolecule required for half maximal binding of the first and second biomolecules.

61. The method according to claim 1 in which 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 a second tether portion or anchor and monitoring any change in levels of bound first and second biomolecules.

64. The method according to claim 1 comprising providing a concentration of first and second biomolecules around the K_d of an interaction between the first and second biomolecules in which the effect of a modulator of the interaction between the first and second biomolecules is determined.

66. Apparatus for determining the affinity of first and second biomolecules by a method according to claim 1 comprising a first biomolecule tethered by a first tether portion having a first tether portion length, a second biomolecule tethered by a second tether portion having a second tether portion length, means for determining binding of adjacent first and second biomolecules to each other, and means for varying at least one of the first and second tether portion lengths.

72. Apparatus according to claim 66 in which the first and second biomolecules are in solution.

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