RNA is a multipotent polymer of great biological and engineering interest. In the last decade, microRNA mimics revolutionized the manipulation of gene expression and are being explored as next-generation therapeutics. Despite their high versatility and specificity, the weak cell-penetrating abilities and rapid endo-/lysosomal degradation pose limitations to their therapeutic applicability. Cell-penetrating peptides (CPPs) are found to be efficient and bio-compatible RNA delivery vectors. Recently, a non-coding RNA antagonist of a prosurvival oncomiR (antagomiR) has been successfully delivered using a pH-responsive amphipathic CPP. Nevertheless, the lack of atomistic information for such systems impedes the rational design and optimization of the vectors. Molecular-level simulations have proven to be very powerful in optimizing small molecule binding to protein targets, and now routinely feature in commercial drug discovery processes. Here, we present our computational results from all-atom medium-to-large-scale simulations on sequence-to-structure modeling and study of the therapeutically relevant antagomiR in complex with amphipathic CPPs. A composite workflow, combining knowledge- and physics-based methods, was developed to fold and sample (exhaustively) relevant states of antagomiR, revealing open- and closed-loop conformations. Separate single- and multi-peptide simulations explored charge-conformation relationships of pH-responsive CPPs, revealing key conformations and oligomeric complexes corresponding to the biochemical environments of interest. Large-scale antagomiR-CPP association/dissociation simulations in explicit solvent, at experimentally-relevant molecular concentrations, pHs, and different RNA:CPP ratios, revealed important interactions and residues contributing to RNA coating and RNA/CPP-CPP complex behavior. Finally, the effect of RNA and peptide crowding on cluster formation was also investigated. Our study contributes to the elucidation of the pH-dependent RNA-peptide interaction mechanism and nanoparticle formation, and may assist in the discovery of CPP designs for tailored antagomiR intracellular translocation.

Modeling peptide-based RNA delivery systems

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