Forget, S.; Stirnemann, G.

Most naturally occurring ribozymes catalyze self-splicing reactions through a 2\'-OH group. Consequently, experimental structures of precatalytic states often require chemical modifications of the 2\'-OH, such as its removal or methylation. However, the impact of these chemical modifications on the active site structure remains largely unexplored, which raises important questions since methylated structures are often taken as being representative of pre-catalytic states. Here, we employ extensive atomistic simulations critically compared to and fine-tuned on experimental data, and we revisit experimental results to show that 2\'-O-methylation critically affects reactant geometries and, therefore, the possible reaction mechanisms inferred from the structures. Our results also challenge the common assumption that 2\'-O-methylation stabilizes the C3\'-endo puckering conformation. Our findings, consistent with recent experimental data on ribosome structure, reveal that this effect is highly sensitive to the local secondary structure and is often overstated. For three investigated small-cleaving ribozymes, the C2\'-endo conformation observed for chemically-modified active site residues through 2\'-O-methylation is not stable upon methyl group removal to obtain the catalytically-relevant hydroxylated state. This suggests that these geometries arise primarily from a combinaison of steric hindrances and electrostatic interactions with the surrounding environment rather than intrinsic conformational preferences of the ribose upon methylation.