Using Programmable Roadblocks to Probe DNA Target Search

Abstract

We study the target search and cleavage mechanisms of type II restriction endonucleases. In our single molecule technique, micron sized beads are tethered with single DNAs in a microfluidic flow cell and imaged using video microscopy. Under low protein concentration, the cleavage time is limited by the target search time. Using this technique, we have demonstrated that the target search strategy of NdeI employs a single 1D scan of 1000bp DNA at low salt concentration, but switches to a search mode requiring multiple short scans at higher salt. To probe the contributions of sliding and hopping to this 1D search, we are programming dCas9 to bind sites flanking the NdeI cognate site in order to block sliding. We have shown that our roadblocks bind specifically to the chosen roadblock sites and that the presence of one or two flanking roadblocks can reduce the target search rate of NdeI at low salt. Our results demonstrate that sliding must play a role in the 1D search mechanism of NdeI.