Fig. 4: Facile development of potent, selective and portable organophosphate sensors.

a, Summary of biosensor screening results for a panel of ten organophosphates. The compounds screened are clustered by similarity (blue indicates more similar) using a distance matrix of pairwise Tanimoto similarity scores, calculated in ChemMine 19. The molecules that yielded hits are shown in bold type; the minimal ligand concentrations required for Y2H signal generation for optimized receptors (Methods) are indicated (Supplementary Fig. 12 shows additional details). b, The optimized PYR1^DIAZI and PYR1^PIRI are high-affinity sensors. Optimized receptors were tested for responses to nanomolar concentrations of diazinon and pirimiphos-methyl, respectively, as evidenced by Y2H assays and receptor-mediated inhibition of HAB1 phosphatase activity in vitro. PYR1^DIAZI (EC₅₀ = 36 nM [32,40]); PYR1^PIRI (EC₅₀ = 58 nM [50,67]). Wild-type PYR1 was used as a control (gray lines). c, PYR1-derived receptors are portable. PYR1^DIAZI and PYR1^PIRI were tested in a protein-fragment complementation system based on split luciferase reconstitution with NLuc^N-PYR1/NLuc^C-HAB1 fusions in yeast (PYR1^DIAZI, EC₅₀ = 24 nM [12,50]; PYR1^PIRI, EC₅₀ = 19 nM [undef, 29]). d,e, PYR1^DIAZI and PYR1^PIRI are selective for their evolved target ligands. d, Y2H (top) and in vitro phosphatase inhibition assays (bottom) were used to profile receptor responses; the receptors no longer bind the native ligand ABA. Pirimiphos-methyl and diazinon were tested 20 nM, ABA, tested at 5,000 nM in Y2H assays. e, Characterization of receptor selectivity using a Z4-PYR1/VP64-ΔN-HAB1 gene activation circuit in the presence of the activating ligands shown (Supporting File 1 shows quantitative analyses of EC₅₀ values). In all cases, the symbol represents the mean, and the error bars show 1 s.d. and may be smaller than the symbol. All data points represent the mean of triplicate data (n = 3), and error bars represent the standard deviation.

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