title: Measuring DNA-protein binding affinity on a single molecule using optical tweezers.
abstract: DNA-protein interactions may be observed on single molecules with a variety of techniques. However, quantifying the binding affinity is difficult and often requires many (∼100) individual events to characterize the interaction. We use a single λ DNA molecule that provides a lattice of binding sites for proteins. Extending and relaxing the tethered molecule reversibly melts DNA, allowing it to be converted between double-stranded (ds) and single-stranded (ss) forms. By monitoring changes in the properties of the DNA as a function of added protein concentration and fitting to binding models, the DNA-protein interaction may be characterized and quantified. As an example, the high mobility group protein HMGB1(box A  +  B) is observed to stabilize dsDNA. Measuring the strength of this effect allows us to determine the equilibrium association constant for HMGB1(box A  +  B) binding to dsDNA.
authors.affiliation: Department of Physics, Northeastern University, Boston, MA, USA.
authors.forename: Micah J
authors.initials: MJ
authors.lastname: McCauley
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D024243:HMGB1 Protein / Q000032:analysis* / Q000737:chemistry; D008863:Microspheres; D052898:Optical Tweezers
chemical_list: D004277:DNA, Single-Stranded; D024243:HMGB1 Protein; D004247:DNA