Fig. 1: ZTP riboswitch single-molecule FRET constructs.

a The ZTP riboswitch aptamer (Δterm, left) formed by paired elements P1-4 bound to ZMP (Z, blue). A Cy3 fluorophore (green sphere) is attached to solvent accessible U32, and a Cy5 fluorophore (red sphere) is placed at the 3′-end of the DNA tether, which is also 5′-end labeled with biotin (gray sphere) for attachment to the streptavidin-coated slide. In the full-length riboswitch (WT, right), the nucleotides colored in red switch between two conformations, one in which a terminator hairpin (PT) or terminator folds and the ligand is not bound. b Cartoon depicting the vectorial folding (VF) assay, in which an engineered helicase Rep-X (orange and green) releases the riboswitch RNA (black) in a 5′-to-3′ direction by translocating on the cDNA strand (gray) and unwinding in a 3′-to-5′ direction. During unwinding, when Rep-X is proximal to Cy3, a temporary increase in Cy3 fluorescence [i.e., protein-induced fluorescence enhancement (PIFE)] is observed.