Determination of Nucleotide–Nucleotide and Nucleotide–Amino Acid Binding Interactions from All-Atom Potential-of-Mean-Force Calculations

Biomolecular condensates emerge from multivalent interactions between proteins and nucleic acids and are frequently modeled by using coarse-grained molecular dynamics simulations. The parametrization of these models critically depends on atomistic data describing the underlying molecular interactions. In this work, we employ all-atom molecular dynamics simulations and potential-of-mean-force (PMF) calculations to investigate the landscape of interactions between RNA nucleotides and protein amino acids. We begin by characterizing nucleotide–nucleotide binding modes through canonical base-pairing analysis, observing notable agreement in the predictions from both the AMBER03ws and CHARMM36 force fields. Further rationalization of different nucleotide–nucleotide interaction modes involves the calculation of PMFs for ribose–ribose, phosphate–phosphate, and RNA tertiary interactions such as G-quadruplex formation. We also examine the effect of salt concentration on these interactions, finding a reduction in electrostatic self-repulsion for phosphate–phosphate binding upon increasing the ionic strength. Expanding our analysis to amino acids, we first benchmark the performance of both AMBER03ws and a99SB-disp force fields for describing pairwise amino acid interactions, and then, we evaluate different nucleotide–amino acid binding profiles. Our findings reveal a subset of amino acids─Lys and Arg (positively charged), Asp and Glu (negatively charged), and Gln, Ser, and Asn (polar residues)─that consistently engage with the nitrogenous bases of different nucleotides. Such binding is primarily mediated by hydrogen bonding and, in some cases, cation−π interactions. Furthermore, we identify strong π–π stacking interactions with aromatic residues and phosphate–Arg contacts as key contributors to condensate cohesion in RNA–protein condensates. Our comprehensive analysis provides a detailed library of nucleotide–amino acid interactions, offering quantitative insights to inform coarse-grained model parametrization and deepening our understanding of condensate self-assembly, nucleic acid recognition, and phase-separation regulation at the submolecular scale.