Rational design of mechanically active RNAs: de novo engineering of functional exoribonuclease-resistant RNAs

Abstract Mechanically active RNAs represent an emerging class of biomolecules whose function derives from resisting molecular forces. Among them, exoribonuclease-resistant RNAs (xr-RNAs) achieve this by folding into a ring-like topology that physically blocks 5’ → 3’ degra-dation. However, despite years of structural insight, the rational design of such mechanically functional RNA devices has remained elusive. Here, we describe a mechanics-aware RNA design approach that enables de novo engineering of functional xrRNAs. We first identify structural determinants of force resistance by perturbing pseudoknot architecture in a model xrRNA and quantifying resulting efficiencies in the stalling of exoribonuclease XRN1. We then implement these rules in a design framework that integrates explicit topological constraints with molecular dynamics-guided optimization. The resulting synthetic xrRNAs reproduce the ring-like architecture and stall exoribonuclease XRN1 with wild-type-like efficiency. Our top-performing constructs exhibit minimal sequence similarity to known xrRNAs and evade detection by covariance models, yet remain fully functional in vitro . Together, our results show that mechanical function can be rationally designed independent of evolutionary ancestry, laying the groundwork for the design of RNA elements that modulate decay and fine-tune the mechanical stability of engineered transcripts.