Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

26-POS Board 26 CONSTANT PH METADYNAMICS SIMULATIONS IN THE STUDY OF RNA OLIGOMERS Tomás F. D Silva 1 ; Giovanni Bussi 1 ; 1 Scuola Internazionale Superiore di Studi Avanzati (SISSA), SBP, Trieste, Italy RNA molecules have a wide range of biological functions due to their highly flexible structures. Their flexibility stems from complex H-bonding networks defined by both canonical and non canonical base pairs. With some non-canonical base pair interactions requiring (de)protonation events to either stabilize or perturb H-bond networks. Constant pH molecular dynamics (CpHMD) methods provide a reliable tool to describe the conformational space of dynamic structures and to obtain robust calculations of pH-dependent properties (i.e. pK a ). However, pH sensitive methods have rarely been explored in the field of nucleic acids, despite growing biological evidence concerning pH regulation of certain motifs’ H-bond networks. In this work, we present an extension of the stochastic CpHMD method to RNA from the standard XOL3 AMBER force field and demonstrate the accuracy of our method to reproduce pK a ’s of RNA oligomers. Poly-U trimers and pentamers with a single central titrable site were characterized for method validation. To tackle their high degrees of freedom, we have integrated a well tempered (wt) metadynamics approach into the CpHMD methodology (CpH-MetaD). The CpH MetaD technique significantly expanded the sampled conformational space, allowing for more robust and accurate estimates of the oligomers’ pK a shifts with respect to the increased phosphate content: 0.5 (A3mer to A5mer); 0.4 (C3mer to C5mer); 0.6 (U3mer to PolyU); 0.5 (G3mer to G5mer). The predicted pK a values – A[3,5]mer: 3.6 (0.1)/4.1 (0.2); C[3,5]mer: 4.6 (0.3) / 5.0 (0.2) ; G[3,5]mer – 10.1 (0.3) / 10.6 (0.2); U[3,5]mer: 9.9 (0.1)/10.5 - and relative shifts are in good agreement with experimental data. Nucleobase stacking and electrostatic interactions with phosphate groups clarify the intramolecular phenomena which dictate the experimentally observed pK a shifts. This work highlights the robustness and accuracy of CpHMD/CpH-MetaD applied to RNA oligomers, and the proton binding affinity sensitivity to phosphate group content in the RNA backbone.

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