Biology subjects

Forget, S.

Publications and source records attributed to Forget, S..

4 recordsLinked to original sources

bioRxiv2026

Mapping Active-Site Conformational Ensembles Along Competing Catalytic Pathways of the Hairpin Ribozyme

The catalytic mechanism of the hairpin ribozyme has remained controversial for more than two decades, with different experimental approaches often supporting distinct mechanistic interpretations. In this work, we investigate the conformational landscape of the active site along several proposed reaction pathways using all-atom molecular dynamics simulations in explicit solvent combined with enhanced sampling techniques. Specifically, we employ Hamiltonian replica exchange simulations to extensively explore active-site conformations without relying on predefined collective variables, enabling a broad characterization of the structural ensembles associated with multiple protonation states along three candidate reaction mechanisms. Our simulations suggest that a dianionic general acid/general base pathway involving direct participation of A38 and G8 is unlikely to proceed through well-defined intermediates with catalytically competent geometries. In particular, states associated with G8 deprotonation and subsequent O2 deprotonation exhibit strongly distorted active-site arrangements that appear poorly suited for reaction progression. Although highly synchronous proton-transfer steps cannot be excluded, the required deprotonation of G8 remains difficult to reconcile with neutral pH conditions. In contrast, monoanionic pathways in which the non-bridging oxygens of the scissile phosphate act as transient proton relays produce intermediates that sample geometries favorable for the nucleophilic addition and leaving-group elimination steps of the reaction. These mechanisms do not require direct catalytic involvement of G8 while remaining compatible with potential acid catalysis by protonated A38+ and a possible structural role of G8. Our results provide a unified conformational perspective on competing mechanistic scenarios. The ensembles generated here offer a foundation for future QM/MM and ML/MM calculations aimed at quantitatively resolving the free-energy landscapes governing hairpin ribozyme catalysis. Finally, the present strategy could easily be applied to other biomolecular systems with high conformational plasticity, including other ribozymes.

biophysics↗

bioRxiv2026

Exploring RNA conformational ensembles in silico: progress and challenges

RNA function is intrinsically linked to its structural polymorphism, with molecules exploring the heterogeneous conformational ensembles resulting from complex energy landscapes. These landscapes arise from competing interactions, small energetic separations between microstates, and strong coupling to the environment, posing significant challenges for both experimental characterization and molecular simulation. In this chapter, we review current computational strategies that aim to explore RNA conformational ensembles in silico, with a specific focus on energy landscape-based approaches and atomistic simulations. We discuss key limitations related to sampling efficiency, force-field accuracy, and ensemble analysis, and illustrate their impact through case studies on a self-cleaving ribozyme and an H-type pseudoknot. Finally, we highlight emerging directions, including closer integration with experimental data and the growing role of machine learning, which will probably reinforce the predictive power of in silico RNA energy landscape exploration.

molecular biology↗

bioRxiv2025

Critical structural perturbations of ribozyme active sites induced by 2'-O-methylation commonly used in structural studies

AO_SCPLOWBSTRACTC_SCPLOWMost 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.

biochemistry↗

bioRxiv2024

Evolving a High-Performance Terminal Deoxynucleotidyl Transferase for Enzymatic DNA Synthesis

Enzymatic DNA synthesis, using stepwise nucleotide addition catalyzed by template-independent polymerases, promises higher efficiency, quality, and sustainability than todays industry standard phosphoramidite-based processes. We report on the directed evolution of a terminal deoxynucleotidyl transferase that uses 3-phosphate blocked dNTPs to control the polymerization reaction and demonstrates high activity for these modified substrates and improved template promiscuity and thermostability.

bioengineering↗