| Autor: |
Kamiya G; Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan., Kitada N; Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan., Furuta T; School of Life Science and Technology, Tokyo Institute of Technology, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan., Thangudu S; Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Palo Alto, California 94304, United States., Natarajan A; Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Palo Alto, California 94304, United States., Paulmurugan R; Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Palo Alto, California 94304, United States., Kim SB; Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan., Maki SA; Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan. |
| Abstrakt: |
Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1-6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue M2 was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure-activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λ max values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with M2 -specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging. |
