A Dynamic Structural Model of Expanded RNA CAG Repeats: A Refined X‑ray Structure and Computational Investigations Using Molecular Dynamics and Umbrella Sampling Simulations

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded CAG repeats can cause Huntington’s and other disease through translation of toxic proteins. Herein, a crystal structure of r­[5′<u>UU</u>GGGC­(C<u>A</u>G)₃GUCC]₂, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that shows both anti–anti and syn–anti orientations for 1 × 1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using AMBER force field in explicit solvent were run for over 500 ns on the model systems r­(5′GCGC<u>A</u>GCGC)₂ (MS1) and r­(5′CCGC<u>A</u>GCGG)₂ (MS2). In these MD simulations, both anti–anti and syn–anti AA base pairs appear to be stable. While anti–anti AA base pairs were dynamic and sampled multiple anti–anti conformations, no syn–anti ↔ anti–anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, an explicit solvent MD simulation over 800 ns was run on r­[5′GGGC­(C<u>A</u>G)₃GUCC]₂, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn–anti conformation), transformed to anti–anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn–anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1 × 1 nucleotide AA internal loops in r­(CAG) repeats is anti–anti but can adopt syn–anti depending on the environment. These results are important to understand RNA dynamic-function relationships and to develop small molecules that target RNA dynamic ensembles