Fig. 1
Design and optimization of a genetically encoded fluorescent sensor of D-2-hydroxyglutarate (D-2-HG). (A) Schematic representation of a FRET-based D-2-HG sensor. The transcription factor DhdR is flanked by two fluorescent proteins (ECFP and cpVenus173) that act as a FRET pair. The structure of DhdR was predicted using AlphaFold and the FRET sensor model was generated in BioRender. The amino acid sequences at the N-terminus and C-terminus of DhdR are shown for the parent sensor and three sensor variants, along with the average Kd’ ± standard deviation for each construct. (B) Representative D-2-HG binding curves for the parent FRET sensor and three sensor variants. Log [D-2-HG] is plotted against the FRET ratio for each construct. (C) Comparison of the dynamic range (Rmax/Rmin) of the parent sensor and three sensor variants. The average dynamic range ± standard deviation is shown for 3–7 independent experiments. Statistical analysis was performed using a one-way ANOVA test with post hoc Tukey (****, P < 0.0001 compared with Parent, Variant 1 and Variant 3; *, P < 0.05 compared with Parent and Variant 1)