Molecular insights on the pre-reactive complex between SHAPE probe and RNA molecules and its role in the SHAPE reaction: a multiscale study

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 understand their biological functions. However, obtaining high-resolution 3D structures remains a significant challenge using 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 calculations on the well characterized GAAA RNA tetraloop, we provide new molecular insights on the pre-reactive complex and its binding mode. Our results highlighted the importance of the local environment in recruiting and adjusting for adequate accommodation of the SHAPE probe.