G-quadruplex modulation by E. coli SSB: A comprehensive study on binding affinities and modes using single-molecule FRET

Abstract

G-quadruplexes (GQs) are essential guanine-rich secondary structures found in DNA and RNA, playing crucial roles in genomic maintenance and stability. Recent studies have unveiled GQs in the intergenic regions of the E. coli genome, suggesting their biological significance and potential as anti-microbial targets. Here, we investigated the interaction between homo-tetrameric E. coli SSB and GQ-forming single-stranded DNA (ssDNA) sequence with varying lengths. Combining Microscale Thermophoresis (MST) and conventional spectroscopic techniques, we explored E. coli SSB binding to ssDNA and the structural changes of these secondary DNA structures upon protein binding. Subsequently, we have utilized smFRET to probe the conformational changes of GQ-ssDNA structures upon SSB binding. Our results provide detailed insights into SSB's access to various GQ-ssDNA sequencies and the wrapping of this homo-tetrameric protein around GQ-ssDNA in multiple distinct binding modalities. This study sheds light on the intricate details of E. coli SSB's interaction with ssDNA and the resulting widespread conformational changes within these oligonucleotide structures after protein binding. It offers a thorough insight into SSB's accesses to various GQ-ssDNA architectures. The finding demonstrates the multifaceted binding methods through which this homo-tetrameric protein envelops GQ-ssDNA and could prove valuable in deciphering biological processes that involve DNA G-quadruplexes.