Abstract
The thrombin binding aptamer (TBA) is a prototypical platform used to understand the impact of chemically-modified nucleotides on aptamer stability and target affinity. To provide structural insight into the experimentally-observed effects of modification size, location, and number on aptamer performance, long time-scale molecular dynamics (MD) simulations were performed on multiple binding orientations of TBA-thrombin complexes that contain a large, flexible tryptophan thymine derivative (T-W) or a truncated analogue (T-K). Depending on modification position, T-W alters aptamer-target binding orientations, fine-tunes aptamer-target interactions, strengthens networks of nucleic acid-protein contacts, and/or induces target conformational changes to enhance binding. The proximity and 5'-to-3' directionality of nucleic acid structural motifs also play integral roles in the behavior of the modifications. Modification size can differentially influence target binding by promoting more than one aptamer-target binding pose. Multiple modifications can synergistically strengthen aptamer-target binding by generating novel nucleic acid-protein structural motifs that are unobtainable for single modifications. By studying a diverse set of modified aptamers, our work uncovers design principles that must be considered in the future development of aptamers containing chemically-modified nucleotides for applications in medicine and biotechnology, highlighting the value of computational studies in nucleic acids research.