Fig. 1: Optimization of the dynamic range, sensitivity, and specificity of the Tango-Trio platform.

a Comparison of TRE and TRE-Tight. Promoters were cloned upstream luc2, and expression vectors were transfected in HEK293T cells along with the β-arrestin2-TEV fusion protein and DRD2. Transfected cells were stimulated with the DRD2 specific agonist quinpirole. b Selection and pharmacological characterization of the monoclonal reporter cell line HTTL (HEK293T-TRE-Tight-Luc2) compared to the original HTL (HEK293T-TRE-Luc) cell line. c Comparison of TEV and TEV219 proteases. β-arrestin2 was cloned to both proteases, and transfected in HTL cells with DRD2. Transfected cells were stimulated with the DRD2 specific agonist quinpirole. HTTL-B2 and HTLA were transfected with HTR2A (e), HTR2B (f), HTR1B (g), and F2R (h) and stimulated, along with untransfected cells (d), with dose-response curve of PMA and in presence/absence of 10 µM JAK inhibitor I. HTTL-B2 and HTLA dose-response curves at various targets: DRD2 to quinpirole (i), HTR5A to serotonin (j), CHRM4 to carbachol (k), OPRM1 to DAMGO (l), ADRB3 to isoproterenol (m), and PTGDR to prostaglandin D2 (n). o–r Comparison of the specificity of HTTL-B2 and HTLA readouts. Cell lines were transfected with GPCRs that activate the Jak/STAT Pathway and stimulated with serial dilutions of untreated FBS (o), heat-inactivated (p), dialyzed (q), and Tet-System Approved (r) sera. HTTL-B2 was maintained in cumate-containing media throughout. Dose- response curves were built using XY analysis for non-linear regression curve and the 3-parameters dose-response stimulation function. Data are presented as mean values, with error bars representing SD. Data are representative of 2 biological replicates, with 3 technical replicates each. Generic receptor codes refer to the GPCR-Tango constructs.