Glycine detection with a nuclease-stable L-RNA sensor

Glycine is a vital extracellular signal in bacteria, plants, and the brain. Although RNA-based sensors detect glycine in cells, their extracellular application in native biological environments is limited by enzymatic degradation from nucleases. Mirror-image RNA is nuclease-resistant and preserves the tertiary structure required for RNA function, but synthesizing long L-RNAs such as the 170-nt glycine sensor (glyS) remains challenging. Here, we applied cross-chiral ligation with natural D-RNA ribozymes to assemble a mirror-image L-RNA glycine sensor (L-glyS). Optimization of the ligation conditions enabled up to 68% conversion to the full-length sensor. L-glyS displayed nuclease resistance and maintained glycine-dependent fluorescence in serum, where the original D-glyS lost function. These results establish cross-chiral ligation as a strategy for constructing long, functional L-RNAs and broaden the possible applications of RNA-based sensors to extracellular detection of small molecules.