Fig. 4. Single-molecule FRET studies of semi-synthetic tbpA aptamers.

a Schematic representing the procedure to obtain the semi-synthetic tbpA aptamer. A 5’ synthetic RNA strand contains Cy3 and Cy5 dyes at the 5’ extremity and position 14, respectively. The complete aptamer is reconstituted by ligating the 5’ strand and a 3’ T7 RNAP-transcribed strand. The aptamer is attached to a PEG slide using a sequence that is hybridized to a DNA anchor containing a biotin. b Ligation reactions performed in the presence of the 5’ and 3’ RNA strands. While the 3’ strand corresponds to the 67 nt product, the ligated product of both RNA molecules corresponds to the 107 nt product. c smFRET histograms of the semi-synthetic tbpA aptamer obtained in the absence and presence of 1 mM TPP. The folded (F) and unfolded (U) states are indicated. Histograms obtained without and with TPP were built using 264 molecules and 323 molecules, respectively. d smFRET time trajectories obtained in the absence (Left) and presence (Right) of TPP. The anti-correlated Cy3 donor and Cy5 acceptor emission intensities are shown with the resulting FRET trace. Photobleaching events are shown by an asterisk. e smFRET contour plots obtained in the absence (Left) and presence (Right) of TPP. Selected traces have been accumulated for the first 50 s. Histograms representing the percentual occupancy of each state are shown to the right. Contour plots obtained without and with TPP were built using 48 molecules and 51 molecules, respectively.