Understanding the photophysics of the Spinach-DFHBI RNA aptamer-fluorogen complex to improve live-cell RNA imaging

The use of aptamer-fluorogen complexes is an emerging strategy for RNA imaging. Despite its promise for cellular imaging and sensing, the low fluorescence intensity of the Spinach-DFHBI RNA aptamer-fluorogen complex hampers its utility in quantitative live-cell and high-resolution imaging applications. Here we report that illumination of the Spinach-fluorogen complex induces photoconversion and subsequently fluorogen dissociation, leading to fast fluorescence decay and fluorogen-concentration-dependent recovery. The fluorescence lifetime of Spinach-DFHBI is 4.0 ± 0.1 ns irrespective of the extent of photoconversion. We detail a low-repetition-rate illumination scheme that enables us to maximize the potential of the Spinach-DFHBI RNA imaging tag in living cells.