Photochemical properties of Spinach and its use in selective imaging

Abstract

The progress in imaging instrumentation and probes has revolutionized the way biologists look at living systems. Current tools enable both observation and quantification of biomolecules, allowing the measurement of their complex spatial organization and the dynamic processes in which they are involved. Here, we report reversible photoconversion in the Spinach system, a recently described fluorescent probe for RNA imaging. Upon irradiation with blue light, the Spinach system undergoes photoconversion to a less fluorescent state and fully recovers its signal in the dark. Through thermodynamic titration, stopped-flow, and light-jump experiments, we propose a dynamic model that accounts for the photochemical behavior of the Spinach system. We exploit the dynamic fluorogen exchange and the unprecedented photoconversion properties in a non-covalent fluorescence turn-on system to significantly improve signal-to-background ratio during long-term microscopy imaging.

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

“…3). Therefore, DFHBI and its associated ions and waters can more readily exchange with bulk solvent, consistent with the photophysical properties of Spinach 6,26,27 .…”

“…The two K + ions are separated by 3.8 Å, as previously seen in other G-quadruplexes 24,25 . The complex topology required for the Spinach G-quadruplex to connect to flanking duplexes on both sides may explain the folding difficulties exhibited by some Spinach sequences 8,26,27 .…”

“…Spinach is swiftly photobleached, but recovers rapidly in the presence of excess DFHBI (ref. 6,26,27). GFP does not photobleach as readily, but once bleached does not recover 27 .…”

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

“…The first of these groups, Wang et al (64), reported that under blue light irradiation, DFHBI undergoes cis-trans photoisomerization, and compared with the more fluorescent Spinach-cis-DFHBI complex, the Spinach-trans-DFHBI complex is threefold less stable and one-third dimmer. To improve the signal-to-background contrast during long-term imaging, an optical lock-in detection (OLID) scheme was proposed for imaging Spinach-based probes (64).…”

“…To improve the signal-to-background contrast during long-term imaging, an optical lock-in detection (OLID) scheme was proposed for imaging Spinach-based probes (64). Han et al (27) further argued that the fast cis-trans photoisomerization induces fast unbinding of the DFHBI from Spinach, and a new ground-state DFHBI can again bind with Spinach to recover the fluorescence.…”