PROJECT SUMMARY/ABSTRACT Nucleic acid therapies provide a promising pathway for the treatment of diseases that are not amenable to target- ing by traditional small molecule drugs. Reduction in toxicity and improvements in delivery and specificity have been achieved through covalent modifications to naturally occurring RNAs, and these advances have recently led to the regulatory approval of several nucleic acid therapies. For example, small interfering RNA (siRNA) silences the expression of disease-causing messenger RNAs (mRNAs). However, this strategy can cause deleterious silencing of off-target mRNAs with partial complementarity to the siRNA guide strand. A strategy for mitigation of off-target silencing is destabilization of base pairing in one end of the duplex by substitution of the siRNA guide strand by glycol nucleic acid (GNA), a nucleic acid analogue with an acyclic backbone. The precise mecha- nisms of many covalent modifications, as well as their dependence on features such as sequence position and structural motifs, are still poorly understood. Physics-based atomistic simulations can interrogate the impact of these modifications on the conformational ensembles of nucleic acids and expedite the trial-and-error process for molecules that are difficult to synthesize in the laboratory. The success of such simulations relies on the accuracy of the force field, a parameterized function that uses classical physics to estimate potential energies from atomic coordinates. A major obstacle to the application of physics-based simulations to therapeutic nucleic acids is the difficulty in developing accurate force field parameters for covalently modified nucleotides. This proposal aims to advance the role of simulations in the design and mechanistic understanding of therapeutic RNAs with covalent modifications by developing an open source workflow to derive force field parameters and run simulations of duplex hybridization for realistic therapeutic siRNAs. In Aim 1, a force field for nucleic acids with arbitrary covalent modifications and backbone chemistry will be parameterized using a systematic and reproducible workflow based on open source software infrastructure maintained by the Open Force Field Initia- tive. In Aim 2, the force field parameters will be validated against experimental measurements of duplex melting temperatures by performing melting simulations for RNA duplexes containing substitutions of GNA. In Aim 3, the weighted ensemble enhanced sampling method will be applied to study the duplex association process for guide siRNAs containing GNA substitutions pairing with target and off-target mRNAs. The proposed research will enable reliable atomistic simulations of therapeutic RNAs, expanding the available toolkit for rational design of such therapies. The systematic workflow for developing and validating nucleic acid force field parameters will be easily generalizable to other covalent modifications beyond those in the siRNAs studied...