Nanomechanics and co-transcriptional folding of Spinach and Mango

Recent advances in fluorogen-binding RNA aptamers known as “light-up” aptamers provide an avenue for protein-free detection of RNA in cells. Crystallographic studies have revealed a G-Quadruplex (GQ) structure at the core of light-up aptamers such as Spinach, Mango and Corn. Detailed biophysical characterization of folding of such aptamers is still lacking despite the potential implications on their in vivo folding and function. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolded in four discrete steps as force is increased to 7 pN and refolded in reciprocal steps upon force relaxation. Binding of DFHBI-1T fluorogen preserved the step-wise unfolding behavior although at slightly higher forces. In contrast, GQ core unfolding in iMangoIII and MangoIV occurred in one discrete step at forces > 10 pN and refolding occurred at lower forces showing hysteresis. Binding of the cognate fluorogen, TO1, did not significantly alter the mechanical stability of Mangos. In addition to K+, which is needed to stabilize the GQ cores, Mg2+ was needed to obtain full mechanical stability of the aptamers. Co-transcriptional folding analysis using superhelicases showed that co-transcriptional folding reduces misfolding and allows a folding pathway different from refolding. As the fundamental cellular processes like replication, transcription etc. exert pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold.