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

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.