Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51.
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Frykholm K, Freitag C, Persson F, Tegenfeldt JO, Granéli A
Anal. Biochem. 443 (2) 261-268 [2013-12-15; online 2013-08-29]
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.
PubMed 23994563
DOI 10.1016/j.ab.2013.08.023
Crossref 10.1016/j.ab.2013.08.023
pii: S0003-2697(13)00407-7