title: Protein-DNA force assay in a microfluidic format.
abstract: The detailed study of protein-DNA interactions is a core effort to elucidate physiological processes, including gene regulation, DNA repair and the immune response. The molecular force assay (MFA) is an established method to study DNA-binding proteins. In particular, high-affinity binder dissociation is made possible by the application of force. Microfluidic lab-on-a-chip approaches have proven helpful for parallelization, small sample volumes, reproducibility, and low cost. We report the successful combination of these two principles, forming a microfluidic molecular force assay and representing a novel use for the established MITOMI chip design. We present, characterize, validate and apply this integrated method. An alternative confocal fluorescence microscopy readout and analysis method is introduced and validated. In a multiplexing application, EcoRI binding is detected and characterized. This method paves the way for quantitative on-chip force measurements. It is suited for integration with DNA micro-spotting and in vitro expression of transcription factors to form a high-throughput chip for detailed DNA-protein interaction studies.
authors.affiliation: Lehrstuhl für Angewandte Physik and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Amalienstrasse 54, 80799 Munich, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Otten
authors.forename: Philip
authors.initials: P
authors.lastname: Wolf
authors.forename: Hermann E
authors.initials: HE
authors.lastname: Gaub
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D014157:Transcription Factors; D004247:DNA; D015246:Deoxyribonuclease EcoRI