Single Molecule Force Measurements Reveal Energy Stabilization of Theophylline Aptamer RNA Switch Required for Gene Control

RNA aptamers play essential roles in gene regulation, sensing, and therapeutics, relying on ligand-induced kinetic changes for function. The theophylline aptamer has been engineered to regulate synthetic gene networks in response to the presence of the theophylline small molecule ligand, but the thermodynamic and kinetic parameters behind theophylline's unique function are not known. Here, we use optical tweezers single-molecule force spectroscopy to measure the folding dynamics of the theophylline aptamer in the presence and absence of ligand. We find that theophylline binding does not affect folding rates but significantly slows unfolding by stabilizing the aptamer by ∼3.6 <i>k</i> _B <i>T</i>, or ∼2.1 kcal/mol. These results support a conformational selection model of ligand binding and reveal no evidence of large structural rearrangement upon ligand association. Our findings suggest that theophylline-induced stabilization, rather than structural remodeling, drives aptamer function in synthetic biology applications to regulate gene expression. This kinetic stabilization supports prior work proposing a kinetic trap mechanism, in which the long-lived, folded ligand-bound state delays ribosome binding and translation. These insights emphasize the kinetic basis of RNA-mediated regulation and inform the future design of ptamer-based tools in synthetic biology and therapeutics.