RNA adapts its flexibility to efficiently fold and resist unfolding.

Recent studies have demonstrated that the mechanisms through which biopolymers like RNA interconvert between multiple folded structures are critical for their cellular functions. A major obstacle to elucidating these mechanisms is the lack of experimental approaches that can resolve these interconversions between functionally relevant biomolecular structures. Here, using a nano-electronic device with microsecond time resolution, we dissect the complete set of structural rearrangements executed by an ultra-stable RNA, the UUCG stem-loop, at the single-molecule level. We show that the stem-loop samples at least four conformations along two folding pathways leading to two distinct folded structures, only one of which has been previously observed. By modulating its flexibility, the stem-loop can adaptively select between these pathways, enabling it to both fold rapidly and resist unfolding. This paradigm of stabilization through compensatory changes in flexibility broadens our understanding of stable RNA structures and is expected to serve as a general strategy employed by all biopolymers.