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

Journal article, 2025

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 ten sets of nanochannels where thousands of DNA molecules can be simultaneously analyzed in different conditions. Using this approach, we investigate Rok, a multifunctional Bacillus subtilis 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 hybridization of 12 bp complementary single-stranded DNA overhangs, suggesting a potential role in homology search during recombination. Rok also displays sequence-selective binding, preferentially associating with 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: A Rok, lacking the neutral part of the internal linker, and sRok, a naturally occurring variant without the linker. This comparison highlights t|he 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.