Analyzing the structural conformation andstability of timP RNA using Optical Tweezers

Abstract

RNA molecules play a central role in gene regulation, where structural dynamics strongly influence their biological function. In particular, pseudoknot formation has been implicated in regulating translation in bacterial toxin-antitoxin systems, including the timP RNA system. In this work, single-molecule optical tweezers (OT) were used to investigate the force-dependent unfolding behavior of timP RNAconstructs, includingwild-type(WT), and two mutants. RNA molecules were tethered in a dual-trap optical tweezers setup and subjected to controlled mechanical stretching while force-extension trajectories were recorded and analyzed. Two distinct unfolding patterns were frequently observed and classified as low force (LF) and high-force (HF) patterns. The HF pattern, characterized by a pronounced unfolding event at higher forces, was associated with the pseudoknot conformation based on the corresponding unfolding of approximately 100 nucleotides. This amount was consistent with the expected size of the pseudoknot structure. The relative occurrence of these HF patterns in the WT system increased with decreased pulling speed and increased refolding time. Similar trends were observed for the M2 and M2M2’ mutants, although the magnitude of the changes varied between constructs. HF trajectories were further analyzed using scatterplot-based clustering of individual force-drop events to extract statistical properties such as unfolding forces and estimated contour length changes. Three reoccurring groups of unfolding events were identified across the investigated constructs, with estimated nucleotide releases broadly corresponding to the expected sizes of structural elements within the timP RNA. Differences in the force and nucleotide distributions were also observed between WT and mutant constructs. These results indicate that both experimental conditions and sequence modifications influence the force-dependent unfolding behavior of timP RNA, while the observed unfolding patters and contour length changes support the association of the high-force pattern with the pseudoknot-containing conformation.