Using HS-AFM to Elucidate M5C-Mediated Intermolecular Dynamics of RNA G-Quadruplex and RNA Aggregation

RNA modification is an indispensable layer in gene regulation. They not only influence the binding of RNA-binding proteins to RNAs but also change many aspects of local RNA fol ding .Multiple lines of evidence demonstrate that RNA modifications can completely alter local RNA properties, including thermal stability, intermolecular interactions, and the bona fide formation of new structures . However, quantitative characterization of the intermolecular interaction remains challenging due to the complexity of in vivo environment. We previously discovered that m5 Cs in RNA G-quadruplex (rG4) can drastically enhance the intermolecular interactions between structural RNAs or promote the formation of intermolecular structures .The exact molecular mechanism is yet to be revealed . To address this knowledge gap, we propose using high-speed atomic force microscopy (HS -AFM) to quantitatively di ssect the formation of intermolecular interactions of RNA oligos with m5C s . In this study, we wi ll use rG4-forming segments derived from human IncRNA MALAT 1 and mouse mRNA KMT2D to quantitatively investigate RNA folding and aggregation under rG4-stabilizing (K+) and destabilizing (Li+) conditions . Given that RNA aggregation may depend on RNA concentration,an RNA concentration gradient will be applied to investigate the underlying influence . HS -AFM will probe the formed RNA oligomers to infer the m5C-induced (or enhanced) RNA structure, followed by multiple spots scanning to statistically identify the dominant structure types in the population. The structural composition captured by HS -AFM at distinct RNA concentrations under rG4 stabilizing and destabilizing conditions wi ll be used to infer the shifted RNA aggregation dynamics influenced by m5C .