Unlocking precision in aptamer engineering: a case study of the thrombin binding aptamer illustrates why modification size, quantity, and position matter

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.