title: Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51.
abstract: Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.
authors.affiliation: Department of Physics, University of Gothenburg, SE-412 96 Gothenburg, Sweden.
authors.forename: Karolin
authors.initials: K
authors.lastname: Frykholm
authors.forename: Camilla
authors.initials: C
authors.lastname: Freitag
authors.forename: Fredrik
authors.initials: F
authors.lastname: Persson
authors.forename: Jonas O
authors.initials: JO
authors.lastname: Tegenfeldt
authors.forename: Annette
authors.initials: A
authors.lastname: Granéli
mesh_terms: D001583:Benzoxazoles / Q000032:analysis* / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008856:Microscopy, Fluorescence; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation; D011806:Quinolinium Compounds / Q000032:analysis* / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*
chemical_list: D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D004247:DNA; D051135:Rad51 Recombinase
keywords: DNA; Fluorescence microscopy; Microfluidics; Rad51; Single molecule; Supported lipid bilayer