title: Semiquantitative and quantitative analysis of protein-DNA interactions using steady-state measurements in surface plasmon resonance competition experiments.
abstract: One method commonly used to characterize protein-DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein; the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested; this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.
authors.affiliation: School of Science and Health, University of Western Sydney, Penrith, NSW 2751, Australia. r.gamsjaeger@uws.edu.au
authors.forename: Roland
authors.initials: R
authors.lastname: Gamsjaeger
authors.forename: Ruvini
authors.initials: R
authors.lastname: Kariawasam
authors.forename: Line H
authors.initials: LH
authors.lastname: Bang
authors.forename: Christine
authors.initials: C
authors.lastname: Touma
authors.forename: Cuong D
authors.initials: CD
authors.lastname: Nguyen
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Matthews
authors.forename: Liza
authors.initials: L
authors.lastname: Cubeddu
authors.forename: Joel P
authors.initials: JP
authors.lastname: Mackay
mesh_terms: D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011506:Proteins; C477058:SSBP2 protein, human; D004247:DNA
keywords: Competition experiments; Kinetics; Protein–DNA interaction; SPR