Photo-Activatable RNA Switches for Optical Control of Cell-Free Protein Synthesis

Abstract Programmable RNA switches enable target-dependent regulation of cell-free protein synthesis. Here, we report a photo-activatable RNA switch using photocleavable Lock DNA (pcl-Lock) to enable optical control of in vitro translation. Synthesized by solid-phase methods, pcl-Lock incorporates a coumarin-based photocleavable linker that maintains the RNA switch in an inactive state. Irradiation with 456 nm light cleaves the linker, releasing inhibition and activating translation. Light-triggered activation produced β-galactosidase, yielding approximately 4-fold and 30-fold increases in fluorescence and glucose signals, respectively. Native polyacrylamide gel electrophoresis confirms efficient photocleavage of the pcl-Lock strand, while control experiments verify that light-dependent activation does not occur with the Lock strand lacking the photocleavable linker. This strategy enables optical regulation of cell-free protein synthesis and establishes a strategy for integrating photochemical control with programmable gene regulation.