Method of measuring the affinity of biomolecules

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 - 74 . (canceled) 
     
     
         75 . An assembly comprising a plurality of tethered biomolecules, each member of said plurality comprising
 a first biomolecule tethered by a first tether portion having a first tether portion length and a first tether persistence length and   a second biomolecule 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 together such that a swept volume is defined for each member of said plurality by the first and second tether portion lengths,   and members of said plurality also having means for determining binding of first and second biomolecules to each other.   
     
     
         76 . The assembly of  claim 75 , wherein the first and second tether portions are provided by a single tether. 
     
     
         77 . The assembly of  claim 75 , wherein at least one member of said plurality is in solution. 
     
     
         78 . A method of measuring the affinity of a first and a second biomolecule comprising
 providing a plurality of tethered biomolecules in solution,   each member of said plurality comprising   a first biomolecule tethered by a first tether portion having a first tether portion length and a first tether persistence length and   a second biomolecule 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 together such that a swept volume is defined for each member of said plurality by the first and second tether portion lengths, 
   and members of said plurality also having means for determining binding of first and second biomolecules to each other;   varying at least one of said first and/or second portion lengths and/or said first and/second persistence lengths; and   measuring the binding of the first and second biomolecules.   
     
     
         79 . The method of  claim 78 , wherein a range of the first and second tether portion lengths produce a range of effective concentrations for the first and/or second biomolecules. 
     
     
         80 . The method of  claim 79 , wherein said method further comprises determining the binding of the first and second biomolecules in the presence of a third biomolecule. 
     
     
         81 . The method of  claim 79 , wherein the length of the first and/or second tether portion are different. 
     
     
         82 . The method of  claim 78 , wherein the at least one tether portion comprises nucleotides. 
     
     
         83 . The method of  claim 78 , wherein the at least one tether portion comprises a double-stranded DNA. 
     
     
         84 . The method of  claim 78 , wherein the at least one tether portion comprises a carbon nanotube, an amyloid fibril, or a polymer. 
     
     
         85 . The method of  claim 84 , wherein the polymer is a DNA crossover complex. 
     
     
         86 . The method of  claim 78 , 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. 
     
     
         87 . The method of  claim 78 , 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. 
     
     
         88 . The method of  claim 78 , wherein the proportion of binding of the first and second biomolecules is determined by the intensity of fluorescence (Forster) resonance energy transfer (FRET) between a first and second fluorophores respectively attached to, or integrated with, the first and second biomolecules. 
     
     
         89 . The method of  claim 78 , 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. 
     
     
         90 . The method of  claim 78 , 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. 
     
     
         91 . The method of  claim 78 , 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. 
     
     
         92 . The method of  claim 78 , wherein the second biomolecule is selected from a library. 
     
     
         93 . The method of  claim 78 , 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. 
     
     
         94 . 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.