Computational Analysis of Sequence Editability in the Theophylline RNA Aptamer as a Functional RNA Module

RNA aptamers are often used as ligand-recognition modules in engineered RNA systems, but integration into larger RNA constructs can influence stability and ligand binding. As a result, aptamer sequences may need to be adapted to new environments while preserving essential properties. Here, we examine this sequence editability problem for the theophylline RNA aptamer. Starting from the experimentally determined structure, we introduced targeted mutations in peripheral structural elements while leaving the recognition site unchanged. The native aptamer, mutated variants, a Mg²⁺-depleted system, and a caffeine-bound control were analyzed using three independent 1 μs molecular dynamics simulations. Binding energetics were estimated with multiple end-point as well as alchemical free energy approaches. Results were interpreted together with base pair stability, the conformational landscape of the binding pocket, and per-nucleotide energy contributions. This allows us to predict whether an edit is tolerated or disruptive. Some mutations retained structural and energetic profiles comparable to the native aptamer, whereas others reduced ligand affinity by propagating structural distortions into the binding pocket. These results show that sequence changes outside the binding site can modulate ligand binding indirectly, and that the ligand interaction network is useful for evaluating edited aptamers. The introduced workflow provides a novel combination of established computational strategies for efficient in silico screening of aptamer variants before experimental testing and can be integrated into the design of larger RNA structures. This works particularly well when an experimental structure is available and the tested mutations are small enough not to disrupt the folding pathway.