Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET

NiCo riboswitch apta-FRET. (A) Secondary structure of the NiCo riboswitch and aptamer insertions (left). Relative organization of NiCo apta-FRET chimera sites upon folding (right). (B) E'_FRET signal resulting from a Co²⁺ titration of construct 27 (see Supplemental Table S1), fit to the Hill equation. (C) E'_FRET observed for each of the NiCo apta-FRET chimeras plotted as a function of the Mango-III, P2 helical extension. The corresponding Broccoli P4 extension is indicated by circles: 0 bp; squares: 1 bp; diamonds: 2 bp; and triangles: 3 bp (see Supplemental Table S1 for the full set of sequences). Closed symbols indicate the presence of 10 µM CoCl₂, open symbols are in the absence of CoCl₂. As a visual aid, data points are connected by smooth curves.