Kinetic Traps in RNA Folding: Targeted Design of Frameshifting Element Mutants by Thermodynamic and Kinetic Analysis of the Chikungunya Virus

Chikungunya virus (CHIKV) employs a programmed ribosomal frameshifting element (FSE) to regulate the synthesis of its viral proteins, making the FSE an attractive antiviral target. Yet the structural dynamics that govern its function are complex and poorly understood, with multiple folds discovered. Through computational analysis, we suggest that the FSE's conformation is determined by a competition between thermodynamic stability and cotranslational folding kinetics. Using an integrated computational pipeline, we map the FSE's equilibrium landscape, revealing a thermodynamically favored pseudoknot that emerges only with sufficient flanking residues. We then use kinetic simulations to show that, for the wildtype sequence, this pseudoknot is often kinetically trapped in simpler, less stable stem loop structures that form more rapidly during synthesis. Using this information, we rationally design several mutants to target different folds in the FSE's repertoire. We demonstrate that while a purely thermodynamic design can fail due to kinetic traps, an iterative design procedure, informed by kinetic analysis, can drive the FSE onto a target conformation. Our work explores conformational plasticity and multiple folding pathways of the CHIKV FSE, shows how cotranslational kinetics influence the fold-switching landscapes, establishes a computational framework for kinetic-based RNA engineering, and highlights the importance of considering folding pathways in the design of RNA-targeted therapeutics.