Affinity series of genetically encoded high sensitivity Förster Resonance Energy Transfer sensors for sucrose

Genetically encoded fluorescent sugar sensors are valuable tools for the discovery of transporters and for quantitative monitoring of sugar steady-state levels in intact tissues. Genetically encoded Forster Resonance Energy Transfer sensors for glucose have been designed and optimized extensively, and a full series of affinity mutants is available for in vivo studies. However, to date, only a single improved sensor FLIPsuc-90{micro}{Delta}1 with a Km for sucrose of [~]90 {micro}M is available for sucrose monitoring. This sucrose sensor was engineered on the basis of an Agrobacterium tumefaciens sugar binding protein. Here, we took a two-step approach to first systematically improve the dynamic range of the FLIPsuc nanosensor and then expand the detection range from micromolar to millimolar sucrose concentrations by mutating a key residue in the binding site. The resulting series of sucrose sensors may allow systematic investigation of sucrose transporter candidates and comprehensive in vivo analyses of sucrose concentration in plants. Since FLIPsuc-90{micro} also detects trehalose in animal cells, the new series of sensors can be used to investigate trehalose transporter candidates and monitor trehalose steady-state levels in vivo as well.