Small Molecule Detection - Engineering Spinach Based Biosensors

Since the discovery of Green Fluorescent Protein, it has revolutionized fluorescent imaging of biomolecules inside cells and animal tissue. A similar fluorescent signal can be obtained with the RNA aptamer Spinach, which also allows direct optical monitoring of RNA in vivo. The qualitative detection of a small molecule is enabled by fusing Spinach with a small molecule aptamer, however, quantitative detection has never been achieved.
Here we show the construction of a modified biosensor system for concentration dependent detection of the metabolite S-adenosylmethionine, SAM. By adjusting the stability of the linker region of the Spinach-SAM construct, the aptamer folding is made dependent on the concentration of the metabolite. An orthogonal aptamer-fluorophore pair termed Mango-TO3 serves as an internal control of RNA transcription levels. Combining several concentration dependent constructs allows us to construct a biosensor with a broadened dynamic range.