A general strategy to red-shift green fluorescent protein based biosensors

Compared to green fluorescent protein (GFP)-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence, and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization, and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid (ncAA), 3-aminotyrosine (aY), to the chromophores of GFP-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the aY-modified biosensors preserved the molecular brightness, dynamic range, and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.