Method

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.

1 . A method of measuring the affinity of first and second biomolecules, wherein a first biomolecule is tethered by a tether portion having a tether portion length to a second biomolecule,
 determining binding of adjacent first and second biomolecules to each other,   varying at least one of the tether portion length and   determining binding of the first and second biomolecules again,
 wherein the first and second biomolecules are tethered together. 
   
     
     
         2 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the first and second tether portions are provided by a single tether. 
     
     
         10 . The method of  claim 1 , wherein the first and second biomolecules are closely adjacent to each other, such that swept volumes defined by the movement of each biomolecule overlap, so that first and second biomolecules may bind to each other. 
     
     
         11 . The method of  claim 1 , wherein 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 of  claim 11 , wherein a range of binding reactions that a third biomolecule affects is determined by determining binding of the first and second biomolecules. 
     
     
         13 . The method of  claim 1 , wherein the tether or tether portion length is of the order of 30 to 12 000 nm. 
     
     
         14 . The method of  claim 13 , wherein the length of the tether portions is from 60 to 6000 nm. 
     
     
         15 . The method of  claim 14 , wherein the length of the tether portions is from 60 to 2000 nm. 
     
     
         16 . The method of  claim 1 , wherein at least one tether portion includes an elongate tether body portion. 
     
     
         17 . The method of  claim 1 , wherein at least one tether portion includes a biomolecule-engaging tether head portion. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein at least one tether portion comprises nucleotides. 
     
     
         20 . The method of  claim 1 , wherein the tether portion comprises double-stranded DNA. 
     
     
         21 . The method of  claim 19 , wherein the tether length is 50 bases to 50 kb. 
     
     
         22 . The method of  claim 21 , wherein the tether length is 200 kb to 20 k bases. 
     
     
         23 . The method of  claim 20 , wherein at least a portion of the, or each, tether portion is produced by translation of an mRNA sequence. 
     
     
         24 . The method of  claim 1 , wherein the tether comprises a carbon nanotube, amyloid fibril or a polymer. 
     
     
         25 . The method of  claim 1 , wherein the polymer is a DNA crossover complex such as a DX hybrid. 
     
     
         26 . The method of  claim 1 , wherein the tether is modulated by chemical modification or physical association. 
     
     
         27 . The method of  claim 26 , wherein the tether portion includes dsDNA, in which the resilience of the tether is modulated by chemical modification or interchelation. 
     
     
         28 . The method of  claim 27 , wherein the dsDNA is interchelated with ethidium bromide. 
     
     
         29 . The method of  claim 18 , wherein the tether is formed by ligating a tether body portion to tether head and tail portions in solution. 
     
     
         30 . The method of  claim 16 , wherein the tether is formed by chemically cross-linking tether head and tail portions to the respective ends of a tether body portion. 
     
     
         31 - 38 . (canceled) 
     
     
         39 . The method of  claim 10 , wherein the swept volumes are each of the order of 2×10 to 1×10 nm. 
     
     
         40 . The method of  claim 1 , wherein the nano- to zeptolitre volumes of the first and/or second biomolecule are used. 
     
     
         41 . The method of  claim 40 , wherein the pico- to attolitre volumes of the first and/or second biomolecule are used. 
     
     
         42 . The method of  claim 1 , wherein 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 of  claim 1 , wherein the proportion of binding of the first and second biomolecules is determined by the intensity of Förster resonance energy transfer (FRET) between the first and second fluorophores respectively attached to, or integrated with, the first and second biomolecules. 
     
     
         44 . The method of  claim 43 , wherein the first fluorophore is <10 nm from the second fluorophore. 
     
     
         45 - 47 . (canceled) 
     
     
         48 . The method of  claim 1 , wherein a pair of first and second biomolecules are used. 
     
     
         49 . The method of  claim 19 , wherein at least one of the first and second biomolecule 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. 
     
     
         50 . The method of  claim 19 , wherein at least one of the first or second biomolecules is a protein or polypeptide chemically cross linked to the tether portion. 
     
     
         51 . The method of  claim 19 , wherein at least one of the first and second biomolecules is a protein or polypeptide/nucleic acid complex generated in situ by annealing a mRNA-DNA conjugate to an immobilized tether and translation extracts to the tethered messenger RNA. 
     
     
         52 . The method of  claim 1 , further comprising a fusion protein between the first or second biomolecule and a second protein domain. 
     
     
         53 . The method of  claim 52 , wherein the second protein domain has a high affinity for the head tether portion. 
     
     
         54 . The method of  claim 1 , wherein the first and/or second biomolecule is a protein, optionally an enzyme, antibody, receptor or a peptide, peptide analogue, a small molecule, polysaccharide a catalytically active RNA species, or a portion thereof. 
     
     
         55 . The method of  claim 43 , wherein the FRET is measured using a confocal microscope, a photomultiplier or TIRF microscope/photomultiplier combination, on glass slides containing arrays of tethered first and second biomolecules. 
     
     
         56 . The method of  claim 42 , wherein the proportion of adjacent first and second biomolecules is indicated by nanoscale spheres or quantum dots attached to or integrated with at least one of the first and second biomolecules. 
     
     
         57 . The method of  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 (K d ) of the first or second biomolecule required for half maximal binding of the first and second biomolecules. 
     
     
         58 . The method of  claim 56 , wherein the affinity of a first biomolecule to a library of second biomolecules is determined. 
     
     
         59 . The method of  claim 58 , wherein the library of biomolecules comprises at least a significant portion of a transcriptome or proteome. 
     
     
         60 . The method of  claim 59 , wherein the library of biomolecules comprises at least 10% of a transcriptome. 
     
     
         61 . The method of  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 tether and monitoring any change in levels of bound first and second biomolecules. 
     
     
         62 . The method of  claim 61 , wherein the tether portion is cleaved enzymatically. 
     
     
         63 . The method of  claim 61 , wherein a photocleavable moiety incorporated in the tether is photo-cleaved to cleave the tether portion. 
     
     
         64 . The method of  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 of the interaction between the first and second biomolecules is determined. 
     
     
         65 . The method of  claim 64 , wherein the modulator is a protein, drug molecule, candidate drug molecule, or mixture of proteins. 
     
     
         66 . Apparatus for determining the affinity of first and second biomolecules by a method according to any preceding claim 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, wherein the first and second biomolecules are tethered together. 
     
     
         67 - 70 . (canceled) 
     
     
         71 . The apparatus of  claim 66 , wherein the first and second tether portions are provided by a single tether. 
     
     
         72 . The apparatus of  claim 66 , wherein the first and second biomolecules are in solution. 
     
     
         73 - 74 . (canceled)