Genetically Encoded Boolean-Responsive FRET Biosensors

Abstract

The development of genetically encoded FRET-based biosensors incorporating Boolean logic operators offers enhanced signal detection abilities within biological complex systems. In this work, we take advantage of autonomously compiled molecular topology, linking FRET donor proteins to accepter species through a series of protease recognition sites with defined connectivity. When intact, the species exhibit FRET; however, when proteolytically separated following user-specified protease input combinations, FRET decreases. Using these methods, we demonstrate a YES-gated reporter sensitive to Sortase 2A9 protease, as well as an AND-gated species that requires both 2A9 and Tobacco Etch Virus (TEV) proteases for complete response. Operation-matched proteolytic cleavage led to a preprogrammed decrease in FRET signaling, confirming input-specific responses. These findings establish a fundamental framework for next-generation biosensors that integrate protein topologies and Boolean logic operators to enhance signal detection with high specificity.