Stimulator of interferon genes (STING) is an endoplasmic reticulum membrane protein that plays a vital role in innate immunity. In this study, we report a new bioorthogonal chemical probe derived from a previously reported synthetic STING agonist by properly installing a trans-cyclooctene group without perturbation of the efficacy of the ligand. The probe is employed to investigate localization dynamics of STING in live cultured human cells and fixed mouse brain tissues. We showed that an optimized labeling strategy employing the probe and tetrazine-silicon rhodamine fluorophore conjugates in a stepwise manner enables the successful tracking of STING in live cells using fluorescence microscopy. Overall, our bioorthogonal approach using the probe will facilitate in-depth investigations of intricate mechanisms associated with STING.
This study was supported in part by the Creative Materials Discovery Program through the National Research Foundation (2019M3D1A1078941), National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2019R1A6A1A11051471, 2021R1C1C1005766, 2021R1C1C1005134, 2022M3E5F3085687, RS-2023-00208819), KIST Institutional Program under Grant (Nos. 2E32825 and 2V10210), Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2022-00166522 GRRC program of Gyeonggi province (GRRCAjou2023-B03) and by the Ajou University research fund. All the chemicals and solvents were purchased from commercial vendors (Alfa Aesar, ThermoFisher Scientific, Tokyo Chemical Industry Co. Ltd. and Sigma-Aldrich) and used without further purification unless otherwise specified. Biomedical grade sodium hyaluronates were purchased from Lifecore Biomedical, Inc. NMR solvents were purchased from Cambridge isotope laboratories, Inc. The reaction progress was monitored via thin-layer chromatography (TLC), and components were visualized by observation under UV light or by treating the TLC plates with stains. 1H and 13C NMR spectra were analyzed using JNM-ECZ 600\\u2009R (JEOL) with chemical shifts reported as ppm (TMS as internal standard). Chemical shifts were reported in parts per million (\\u03B4) and calibrated using internal standard tetramethylsilane (TMS) or residual undeuterated solvent for 1H NMR spectra (CDCl3 7.26\\u2009ppm; CD3OD 3.31\\u2009ppm; DMSO-d6 2.50\\u2009ppm) and 13C NMR spectra (CDCl3 77.00 ppm; CD3OD 49.00\\u2009ppm; DMSO-d6 39.50\\u2009ppm). Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); m (multiplet); dd (doublet of doublet); ddd (doublet of doublet of doublet); td (triplet of doublet); dt (doublet of triplet); etc. Coupling constants were reported in Hz. A compact mass spectrometer (Advion) was used as low-resolution mass spectrometry (LR-MS).
