Rapid discovery and evolution of nanosensors containing fluorogenic amino acids

Binding-activated optical sensors are powerful tools for imaging, diagnostics, and biomolecular sensing. However, the discovery of new biosensors is slow and requires tedious steps in rational design, screening, and characterization. Here we report a platform to streamline biosensor discovery and to unlock directed nanosensor evolution with genetically encodable fluorogenic amino acids (FgAAs). Building on the classical semisynthetic approach, we engineered ~15 kDa nanosensors that recognize specific proteins, peptides, and small molecules with up to 100-fold fluorescence increases and subsecond kinetics, allowing real-time and wash-free target sensing and live-cell bioimaging. Furthermore, an optimized genetic code expansion chemistry with FgAAs allowed cell-free translation of functional nanosensors to enable the characterization of hundreds of candidates in parallel. This capability enabled us to identify improved nanosensors with an unbiased and rapid (~ 3 h) ribosomal discovery approach, and to establish a directed nanosensor evolution pipeline that selected for variant-specific nanosensors with improved sensitivities (up to ~250-fold) for SARS-CoV-2 antigens. Altogether, this platform will accelerate the discovery of fluorogenic nanosensors and pave the way to modify proteins with other non-standard functionalities for diverse applications.