title: Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions.
abstract: Complexity and heterogeneity are common denominators of the many molecular events taking place inside the cell. Single-molecule techniques are important tools to quantify the actions of biomolecules. Heterogeneous interactions between multiple proteins, however, are difficult to study with these technologies. One solution is to integrate optical trapping with micro-fluidics and single-molecule fluorescence microscopy. This combination opens the possibility to study heterogeneous/complex protein interactions with unprecedented levels of precision and control. It is particularly powerful for the study of DNA-protein interactions as it allows manipulating the DNA while at the same time, individual proteins binding to it can be visualized. In this work, we aim to illustrate several published and unpublished key results employing the combination of fluorescence microscopy and optical tweezers. Examples are recent studies of the structural properties of DNA and DNA-protein complexes, the molecular mechanisms of nucleo-protein filament assembly on DNA and the motion of DNA-bound proteins. In addition, we present new results demonstrating that single, fluorescently labeled proteins bound to individual, optically trapped DNA molecules can already be tracked with localization accuracy in the sub-10 nm range at tensions above 1 pN. These experiments by us and others demonstrate the enormous potential of this combination of single-molecule techniques for the investigation of complex DNA-protein interactions.
authors.affiliation: Institute for Lasers, Life and Biophotonics Amsterdam and Department of Physics and Astronomy, VU University Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Andrea
authors.initials: A
authors.lastname: Candelli
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
authors.forename: Erwin J G
authors.initials: EJ
authors.lastname: Peterman
mesh_terms: D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D044085:Microfluidics; D008856:Microscopy, Fluorescence; D052898:Optical Tweezers; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D004247:DNA; D051135:Rad51 Recombinase