Figure 2. The input stem of the F30 three-way junction regulates Broccoli fluorescence on the output stem.

(A) A twelve-nucleotide loop (orange) was inserted on the input stem of the F30 three-way junction to prevent any stabilization effect of the input stem on the output stem. The length of the output stem was varied until Broccoli was destabilized. (B) Six different output stem variants were tested, and Broccoli alone was used as a positive control. RNA (0.5 μM) was incubated with 10 μM DFHBI-1T. Shown is the fluorescence (Ex 460 nm, Em 505 nm) normalized to Broccoli fluorescence. The mean and SEM values are shown (n=3). (C) Metastable output stems from (B) were assessed for fluorescence recovery when the loop was replaced with a 12 bp stable input stem (purple). The Broccoli aptamer was attached to the output stem of the F30 using variants 3-6 from (B). (D) Allosteric activation of Broccoli by the input stem. Broccoli was tested using four different output stem variants. RNA (0.5 μM) was incubated with 10 μM DFHBI-1T and the fluorescence was normalized to Broccoli fluorescence. The fluorescence of each variant is shown in the context of a stable input stem (+) or a loop in place of the input stem (−). The mean and SEM values are shown (n=3). *** P = 0.0002 (3), *** P = 2X10⁻⁶ (4), *** P = 4X10⁻⁸ (5), *** P = 4X10⁻⁷ (6).