Fig. 1: Development and in vitro characterization of HypnoS.
a Schematic of the genetically-encoded iAdo sensor, HypnoS. The N-terminal peptide (RSET) with a His tag originates from the bacterial expression vector pET28a. The X-ray crystal structures of Ado-bound PvADA (PDB: 2PGF) and cpEGFP (PDB: 3WLD) are shown as cartoon with Ado and chromophore of cpEGFP shown as stick and sphere, respectively. The loop bearing the insertion site in PvADA is marked in red. b The top panel outlines the strategy for creating iAdo sensors. The bottom panel shows the responses of various sensor candidates to 100 μM Ado (relative to HypnoS’s response). c One-photon Excitation and emission spectra of Ado-free and Ado-bound HypnoS in test buffer. AU, arbitrary units. d Two-photon excitation spectra of HypnoS. Data were collected from HEK293T cells expressing HypnoS. n = 30 cells for 0 μM Ado and n = 21 cells for 100 μM Ado, respectively. AU, arbitrary units. e Fluorescence response of HypnoS and HypnoS-mut to Ado. The fluorescence response (F488/F398) of HypnoS and a control sensor (HypnoS-mut) upon excitation at 488 nm and 398 nm is plotted against increasing Ado concentrations (n = 3). Emission was measured at 513 nm. f Binding kinetics of HypnoS to Ado. Each curve represents a different Ado concentration (bottom to top: 0.5, 1, 5, 10, 50, and 100 μM). g Nucleotide selectivity of HypnoS. The responses of HypnoS to various nucleotides (n = 3) are shown. Ado (adenosine), Ade (adenine), cAMP (cyclic adenosine monophosphate), AMP (adenosine monophosphate), ADP (adenosine diphosphate), ATP (adenosine triphosphate), Ino (inosine), IMP (inosine monophosphate), Guo (guanosine), GMP (guanosine monophosphate), GDP (guanosine diphosphate), GTP (guanosine triphosphate), Thd (thymidine), Urd (uridine), Cyd (cytidine), NAD+ (nicotinamide adenine dinucleotide), NADH (reduced NAD+), NADP+ (nicotinamide adenine dinucleotide phosphate), and NADPH (reduced NADP+) were added to the sensor solution. h Adenosine deaminase enzyme activity tests (n = 3). PvADA, HypnoS, or HypnoS-mut (1 μM) were added to test buffer containing 100 μM Ado. The change in light absorbance at 265 nm (Abs265 nm) was monitored to track Ado concentration changes. Representative images (left, scale bar 20 μm), the time course (middle, F488/F405) and a summary of the peak ΔR/R0 (right) for HypnoS and HypnoS-mut fluorescence before and after Ado (100 μM, i) or 5-Iodotubercidin (5-ITu, 5 μM, j) application in HEK293T cells. The fluorescence response (HypnoS and HypnoS-mut) is presented as a pixel-by-pixel ratio between the images obtained with 488 nm and 405 nm excitation (F488/F405). n = 75 (HypnoS) and 67 (HypnoS-mut) cells from 3 cultures in i and n = 97 (HypnoS) and 82 (HypnoS-mut) cells from 3 cultures in j. Two-tailed Student’s t-test: in i P = 2.6 × 10−130; in j P = 4.1 × 10−75 between HypnoS and HypnoS-mut. All the data are shown as mean ± s.e.m. with the error bars or shaded regions indicating the s.e.m. ***P < 0.001. Source data are provided as a Source Data file.
