Essential Considerations for Free Energy Calculations of RNA-Small Molecule Complexes: Lessons from Theophylline-Binding RNA Aptamer

Alchemical free energy calculations are widely used to predict the binding affinity of small molecule ligands to protein targets; however, the application of these methods to RNA targets has not been deeply explored. We systematically investigated how modeling decisions affect the performance of absolute binding free energy calculations for a relatively simple RNA model system: theophylline-binding RNA aptamer with theophylline and five analogs. The goal of this investigation was twofold: (1) understanding the performance levels we can expect from absolute free energy calculations for a simple RNA complex and (2) learning about practical modeling considerations that impact the success of RNA binding predictions, which may be different than the best practices established for protein targets. We learned that magnesium ion (Mg ²⁺ ) placement is a critical decision that impacts affinity predictions. When information regarding Mg ²⁺ positions is lacking, implementing RNA backbone restraints is an alternative way of stabilizing RNA structure that recapitulates prediction accuracy. Since mistakes in Mg ²⁺ placement can be detrimental, omitting magnesium ions entirely and using RNA backbone restraints is attractive as a risk-mitigating approach. We found that predictions are sensitive to modeling experimental buffer conditions correctly, including salt type and ionic strength. We explored the effects of sampling in the alchemical protocol, choice of the ligand force field (GAFF2/OpenFF Sage), and water model (TIP3P/OPC) on predictions, which allowed us to give practical advice for the application of free energy methods to RNA targets. By capturing experimental buffer conditions and implementing RNA backbone restraints, we were able to compute binding affinities accurately (MAE = 2.2 kcal/mol, Pearson's correlation coefficient = 0.9, Kendall's tau = 0.7). We believe there is much to learn about how to apply free energy calculations for RNA targets and how to enhance their performance in prospective predictions. This study is an important first step for learning best practices and special considerations for RNA-ligand free energy calculations. Future studies will consider increasingly complicated ligands and diverse RNA systems and help the development of general protocols for therapeutically relevant RNA targets.