Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
Artikel i vetenskaplig tidskrift, 2013

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.

GENERATION

MECHANISMS

DNA

SUPPORTED LIPID-BILAYERS

DIFFUSION

Supported lipid bilayer

Microfluidics

GRADIENTS

HOMOLOGOUS RECOMBINATION

Single molecule

Fluorescence microscopy

Rad51

STRANDED-DNA

FILAMENTS

Författare

Karolin Frykholm

Göteborgs universitet

Camilla Freitag

Göteborgs universitet

Fredrik Persson

Göteborgs universitet

J. O. Tegenfeldt

Göteborgs universitet

Annette Graneli

Göteborgs universitet

Analytical Biochemistry

0003-2697 (ISSN) 1096-0309 (eISSN)

Vol. 443 261-268

Ämneskategorier

Biokemi och molekylärbiologi

DOI

10.1016/j.ab.2013.08.023