Figure 1.

Modification of original Spinach and sensor design. (A) Structure of the original Spinach and the modified Spinach (modSpinach) drawn according to Huang et al. (18). There are three paired regions, P1, P2 and P3. The two junctions between the paired regions are J1–2 and J2–3 (17). The G’s from the G-quadruplex are highlighted in red. A’s and U’s from the same region are highlighted in gold. DFHBI (green) binds between the G’s comprising the top G-quartet and the Hoogsteen-paired (blue) U and A of the base triple of J2–3. To create modSpinach, P1 was closed and P3 was opened and replaced with the 24–2 min Spinach sequence. The bases colored gray were used to stabilize the modSpinach, but not used in the sensor designs. The crucial G-quadruplex and base triple required for DFHBI binding were unaltered in the modSpinach. (B) Fluorescence emission spectra of modSpinach was nearly identical to the original Spinach and was obtained from spectral confocal micrographs (lower panels). The water control shows the background level of free, unbound DFHBI. (C) Overview diagram of the FASTmiR sensor design. The FASTmiR sensor relies on a sxRNA switch that has both an OFF-state and ON-state. The DFHBI binding regions consisting of the G-guadruplex (orange) and the base triple (blue) are disrupted in the OFF-state. The small RNA binding regions (purple) form toe-hold-like site(s) (unpaired) that allow the small RNA target (green) to initiate binding. This potentially destabilizes the OFF-state which allows the FASTmiR sensor to switch (middle panel) to the ON-state (right panel). Once in the ON-state, the DFHBI binding pocket reforms and then fluoresces. The switch may happen also happen independently of the small RNA, but the ON-state is only stabilized when bound by the target small RNA via a three-way junction (3WJ). The ON-state also depicts the three modular components of FASTmiR sensors: the small RNA binding regions, the base of the 3WJ (gray) and the modSpinach.