Real-Time Monitoring of the Multistate Conformational Dynamics of Polypurine Reverse Hoogsteen Hairpin To Capture Their Triplex-Affinity for Gene Silencing by smFRET Microspectroscopy

摘要

Single-molecule Forster resonance energy transfer microspectroscopic methods are employed for real-time monitoring and to gain deeper insights into the formation of the polypurine reverse Hoogsteen hairpin (PPRH) and its triplex-forming activity. The heterogeneity in the behavior of individual PPRHs has been documented, and it is seen that the degree of anharmonic plasticity of the antiparallel hairpin is stabilized by the formation of reverse Hoogsteen (RH) bonds. While being involved in the hairpin formation, they flip reversibly between the open and closed conformations, irrespective of the concentration of ions present in their microenvironment. However, the nature of the cation present in the buffer plays a crucial role in determining the structural stability. The Watson Crick (WC) bonds are found to be more dynamic in the triplex compared to that of the RH base pairs, indicating the involvement of progressive WC bonds during the triplex motif formation by the PPRH. The majority of the intact triplex DNA attained a semifolded relaxed state before progressing toward a tightly folded state, emphasizing the fact that the folding mechanism pursues an ambiguous path in the mode of acquiring the final step of the triple helix motif. Moreover, the presence of triplex-forming sequences in the regulatory regions of the genome further provides an intricate link between the experimental results and sequence occurrence.