Computational Modeling of RNA Aptamers: Structure Prediction of the Apo State

[Image: see text] RNA aptamers are single-stranded oligonucleotides that bind to specific molecular targets with high affinity and specificity. To design aptamers for new applications, it is critical to understand the ligand binding mechanism in terms of the structure and dynamics of the ligand-bound and apo states. The problem is that most of the NMR or X-ray crystal structures available for RNA aptamers are for ligand-bound states. Available apo state structures, mostly characterized by crystallization under nonphysiological conditions or probed by low resolution techniques, might fail to represent the diverse structural variations of the apo state in solution. Here, we develop an approach to obtain a representative ensemble of apo structures that are based on in silico RNA 3D structure prediction and in vitro experiments that characterize base stacking. Using the neomycin-B aptamer as a case study, an ensemble of structures for the aptamer in the apo (unbound) state are validated and then used to investigate the ligand-binding mechanism for the aptamer in complex with neomycin-B.