AbstractThe chemical properties and biological mechanisms of RNAs are determined by their tertiary structures. Exploring the tertiary structure folding processes of RNA enables us to understand and control its biological functions. Here, we report a nanopore snapshot approach combined with coarse-grained molecular dynamics simulation and master equation analysis to elucidate the folding of an RNA pseudoknot structure. In this approach, single RNA molecules captured by the nanopore can freely fold from the unstructured state without constraint and can be programmed to terminate their folding process at different intermediates. By identifying the nanopore signatures and measuring their time-dependent populations, we can “visualize” a series of kinetically important intermediates, track the kinetics of their inter-conversions, and derive the RNA pseudoknot folding pathway. This approach can potentially be developed into a single-molecule toolbox to investigate the biophysical mechanisms of RNA folding and unfolding, its interactions with ligands, and its functions.