High-throughput single-molecule nanofluidic studies on <i>B. subtilis</i> Rok protein interaction with DNA

Single-molecule methods offer powerful insights into DNA-protein interactions at the individual DNA molecule level. We developed an automated, high-throughput nanofluidic imaging platform to characterize DNA-protein complexes in solution. The platform uses a nanofluidic chip with 10 sets of nanochannels where thousands of DNA molecules can be simultaneously analyzed in different conditions. Using this approach, we investigate Rok, a multifunctional <i>Bacillus subtilis</i> protein involved in genome organization and transcription regulation. Our findings confirm the DNA-condensing activity of Rok, likely attributed to its ability to bridge distant DNA segments. Additionally, Rok promotes the hybridization of 12 base complementary single-stranded DNA overhangs, suggesting a potential role in homology search during recombination. Rok also displays sequence-selective binding, preferentially associating with adenine and thymine-rich (AT-rich) DNA regions. To explore the structural features of Rok underlying these activities and test our nanofluidic system further, we compare wild-type Rok with two variants: ∆Rok, lacking the neutral part of the internal linker, and sRok, a naturally occurring variant without the linker. This comparison highlights the role of the linker in hybridization, i.e., interaction with single-stranded DNA. Together, these findings enhance our understanding of Rok-mediated DNA dynamics and establish single-molecule nanofluidics as a powerful tool for high-throughput studies of DNA-protein interactions.