Visualizing intracellular glycine with two-dye and single-dye ratiometric RNA-based sensors

This experiment contains all flow cytometry data from the manuscript "Visualizing intracellular glycine with two-dye and single-dye ratiometric RNA-based sensors." Glycine is an important metabolite and cell signal in diverse organisms, yet tools to visualize intracellular glycine dynamics have not been developed. In this study, diverse and bright RNA-based glycine biosensors were developed by fusing the architecturally complex glycine riboswitch with Broccoli class fluorogenic aptamers. The brightest sensor with the highest activation, glyS, and its two-dye ratiometric counterpart Pepper-glyS, allowed for visualization of a drug-induced accumulation of endogenous glycine in live E. coli cells. However, a general limitation of two-dye orthogonal aptamer pairs is that differences in dye properties may prevent accurate quantitation of cellular targets. Here, a novel DFHO-binding Golden Broccoli aptamer was developed that is easily resolved from Red Broccoli-DFHO by unmixing protocols. This enabled the generation of the first single-dye ratiometric sensor, which detects glycine both in vitro and in live E. coli cells.
Notes:
See the manuscript for experimental details on data acquisition and processing. Each file is labeled with the corresponding manuscript figure, a sample name, and a terminal number indicating the replicate number. See manuscript Methods.