A Multiscale Study to Characterize SHAPE Probe/RNA Molecules Prereactive Complex and Its Role in Initiating the SHAPE Reaction.

Ribonucleic acid (RNA) molecules play a crucial role in nearly every cellular process, with their function closely tied to their three-dimensional (3D) structure. As a result, determining the precise 3D structure of RNAs is essential to understanding their biological functions. However, obtaining high-resolution 3D structures remains a significant challenge with traditional biophysical techniques. To address this, chemical probing methods such as SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) have gained widespread popularity. SHAPE reactivities have been introduced in 2D RNA predictors as soft constraints on nucleotide base pairing, although the acylation reaction involves the ribose moiety. Little is known about the physical chemistry behind this reaction, leaving several reactivities unexplained. In this context, our aim is to unveil the complex relationship between the local structure of RNA, its dynamics, and the SHAPE chemical reactivity. In this study, using a multiscale approach based on biased molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations on the well characterized GAAA RNA tetraloop, we provide new molecular insights on the prereactive complex and its binding mode. Our findings emphasize the critical role of the local environment in facilitating the recruitment and proper accommodation of the SHAPE probe. All-atom umbrella sampling (US) molecular dynamics (MD) simulations underscored the significance of the binding angle in forming the prereactive complex, which was thoroughly analyzed using geometrical descriptors. In US QM/MM simulations, we used different initial structures to demonstrate the effect of favorable binding on the acylation reaction by characterizing the first step. Moreover, we investigated the deprotonation of the hydroxyl and its role in the acylation reaction, highlighting the role of the local environment and the probe proximity in this process. Our results suggest that the formation of oxyanion prior to the binding is not required to initiate the chemical reaction.