Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51

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Frykholm, Karolin,1977Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg (författare)

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. (C) 2013 Elsevier Inc. All rights reserved.

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
NATURVETENSKAP Fysik Den kondenserade materiens fysik hsv//swe
NATURAL SCIENCES Physical Sciences Condensed Matter Physics hsv//eng
Single molecule
DNA
Rad51
Microfluidics
Supported lipid bilayer
Fluorescence microscopy
SUPPORTED LIPID-BILAYERS
HOMOLOGOUS RECOMBINATION
STRANDED-DNA
GRADIENTS
MECHANISMS
GENERATION
DIFFUSION
FILAMENTS
GENERATION

Freitag, CamillaGothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg (författare)
Persson, Fredrik,1979Uppsala universitet,Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg,Beräknings- och systembiologi(Swepub:uu)frepe558 (författare)
Tegenfeldt, Jonas O.Lund University,Lunds universitet,Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),Fasta tillståndets fysik,Fysiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Solid State Physics,Department of Physics,Departments at LTH,Faculty of Engineering, LTH,University of Gothenburg(Swepub:lu)ftf-jte (författare)
Graneli, Annette,1973Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg(Swepub:gu)xgrane (författare)
Göteborgs universitetInstitutionen för fysik (GU) (creator_code:org_t)

Av författaren/redakt...: Frykholm, Karoli ...; Freitag, Camilla; Persson, Fredrik ...; Tegenfeldt, Jona ...; Graneli, Annette ...

Om ämnet

Av lärosätet: Göteborgs universitet; Lunds universitet; Chalmers tekniska högskola; Uppsala universitet

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