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 Escherichia colicells. 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 Golden Broccoli aptamer was developed that is readily resolved from Red Broccoli by spectral unmixing methods, even though both aptamers bind the same dye, DFHO. This enabled generation of the first single-dye ratiometric sensor, which detects glycine both in vitro and in live E. coli cells.