{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:01:18Z","timestamp":1760148078452,"version":"build-2065373602"},"reference-count":26,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2023,3,27]],"date-time":"2023-03-27T00:00:00Z","timestamp":1679875200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Society for the Promotion of Science","award":["21H04948","20K21851","17H01215"],"award-info":[{"award-number":["21H04948","20K21851","17H01215"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Imaging protein\u2013protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.<\/jats:p>","DOI":"10.3390\/s23073498","type":"journal-article","created":{"date-parts":[[2023,3,27]],"date-time":"2023-03-27T05:34:30Z","timestamp":1679895270000},"page":"3498","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Bright Molecular Strain Probe Templates for Reporting Protein\u2013Protein Interactions"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7033-9056","authenticated-orcid":false,"given":"Sung-Bae","family":"Kim","sequence":"first","affiliation":[{"name":"Environmental Management Research Institute (EMRI), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8569, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4568-9043","authenticated-orcid":false,"given":"Tadaomi","family":"Furuta","sequence":"additional","affiliation":[{"name":"School of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Genta","family":"Kamiya","sequence":"additional","affiliation":[{"name":"Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu 182-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3104-6963","authenticated-orcid":false,"given":"Nobuo","family":"Kitada","sequence":"additional","affiliation":[{"name":"Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu 182-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7155-4738","authenticated-orcid":false,"given":"Ramasamy","family":"Paulmurugan","sequence":"additional","affiliation":[{"name":"Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Palo Alto, CA 94304, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9741-0166","authenticated-orcid":false,"given":"Shojiro A.","family":"Maki","sequence":"additional","affiliation":[{"name":"Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu 182-8585, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,27]]},"reference":[{"unstructured":"Shimomura, O. (2006). Bioluminescence, World Scientific Publishing Co., Pte. Ltd.","key":"ref_1"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"233","DOI":"10.2116\/analsci.20R003","article-title":"Bioluminescent Imaging Systems for Assay Developments","volume":"37","author":"Kim","year":"2021","journal-title":"Anal. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1021\/ac3031724","article-title":"Advances in Fluorescence and Bioluminescence Imaging","volume":"85","author":"Ozawa","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2646","DOI":"10.7150\/thno.32219","article-title":"Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo","volume":"9","author":"Nishihara","year":"2019","journal-title":"Theranostics"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1039\/C9CC07634D","article-title":"Ligand-activated BRET9 imaging for measuring protein-protein interactions in living mice","volume":"56","author":"Kim","year":"2020","journal-title":"Chem. Commun."},{"doi-asserted-by":"crossref","unstructured":"El Khamlichi, C., Reverchon-Assadi, F., Hervouet-Coste, N., Blot, L., Reiter, E., and Morisset-Lopez, S. (2019). Bioluminescence Resonance Energy Transfer as a Method to Study Protein-Protein Interactions: Application to G Protein Coupled Receptor Biology. Molecules, 24.","key":"ref_6","DOI":"10.3390\/molecules24030537"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2987","DOI":"10.1021\/acs.jproteome.9b00154","article-title":"Applications of Protein Fragment Complementation Assays for Analyzing Biomolecular Interactions and Biochemical Networks in Living Cells","volume":"18","author":"Li","year":"2019","journal-title":"J. Proteome Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1016\/S0076-6879(10)70014-8","article-title":"A Toolkit of Protein-Fragment Complementation Assays for Studying and Dissecting Large-Scale and Dynamic Protein Protein Interactions in Living Cells","volume":"470","author":"Michnick","year":"2010","journal-title":"Meth. Enzymol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1021\/ac0484777","article-title":"Firefly luciferase enzyme fragment complementation for imaging in cells and living animals","volume":"77","author":"Paulmurugan","year":"2005","journal-title":"Anal. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4820","DOI":"10.1021\/ac0621571","article-title":"Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferase","volume":"79","author":"Kim","year":"2007","journal-title":"Anal. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5990","DOI":"10.1038\/s41598-018-24278-2","article-title":"A Firefly Luciferase Dual Color Bioluminescence Reporter Assay Using Two Substrates To Simultaneously Monitor Two Gene Expression Events","volume":"8","author":"Branchini","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/j.ymeth.2003.10.010","article-title":"Application of the split-ubiquitin membrane yeast two-hybrid system to investigate membrane protein interactions","volume":"32","author":"Fetchko","year":"2004","journal-title":"Methods"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2324","DOI":"10.1021\/bc900330w","article-title":"A molecular tension-indexed bioluminescent probe for determining protein\u2013protein interactions","volume":"20","author":"Kim","year":"2009","journal-title":"Bioconjugate Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1021\/acs.bioconjchem.5b00421","article-title":"Genetically Encoded Molecular Tension Probe for Tracing Protein\u2014Protein Interactions in Mammalian Cells","volume":"27","author":"Kim","year":"2016","journal-title":"Bioconjugate Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"71","DOI":"10.2116\/analsci.18SDP08","article-title":"In vitro Determination of Rapamycin-triggered FKBP-FRB Interactions Using a Molecular Tension Probe","volume":"35","author":"Kim","year":"2019","journal-title":"Anal. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"908","DOI":"10.1038\/s41598-017-00955-6","article-title":"Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays","volume":"7","author":"Nishihara","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2067","DOI":"10.1021\/bc400411h","article-title":"Creation of artificial luciferases for bioassays","volume":"24","author":"Kim","year":"2013","journal-title":"Bioconjugate Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1016\/j.bbrc.2014.04.128","article-title":"Functional artificial luciferases as an optical readout for bioassays","volume":"448","author":"Kim","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/s41929-022-00895-z","article-title":"Catalytic mechanism for Renilla-type luciferases","volume":"6","author":"Schenkmayerova","year":"2023","journal-title":"Nat. Catal."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1126\/science.273.5272.239","article-title":"Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP","volume":"273","author":"Choi","year":"1996","journal-title":"Science"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold: Making protein folding accessible to all","volume":"19","author":"Mirdita","year":"2022","journal-title":"Nat. Methods"},{"doi-asserted-by":"crossref","unstructured":"Kamiya, G., Kitada, N., Furuta, T., Hirano, T., Maki, S.A., and Kim, S.B. (2023). S-Series Coelenterazine-Driven Combinatorial Bioluminescence Imaging Systems for Mammalian Cells. Int. J. Mol. Sci., 24.","key":"ref_22","DOI":"10.3390\/ijms24021420"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1177\/1087057104271865","article-title":"Homogeneous, bioluminescent protease assays: Caspase-3 as a model","volume":"10","author":"Daily","year":"2005","journal-title":"J. Biomol. Screen."},{"doi-asserted-by":"crossref","unstructured":"Kamiya, G., Kitada, N., Furuta, T., Hirano, T., Maki, S., and Kim, S.B. (2022). C-Series Coelenterazine-Driven Bioluminescence Signature Imaging. Int. J. Mol. Sci., 23.","key":"ref_24","DOI":"10.3390\/ijms232113047"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"12288","DOI":"10.1073\/pnas.0404041101","article-title":"Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals","volume":"101","author":"Luker","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4715","DOI":"10.1021\/ja043277y","article-title":"Characterization of the FKBP center dot Rapamycin center dot FRB ternary complex","volume":"127","author":"Banaszynski","year":"2005","journal-title":"J. Am. Chem. Soc."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/23\/7\/3498\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:03:57Z","timestamp":1760123037000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/23\/7\/3498"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,27]]},"references-count":26,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["s23073498"],"URL":"https:\/\/doi.org\/10.3390\/s23073498","relation":{},"ISSN":["1424-8220"],"issn-type":[{"type":"electronic","value":"1424-8220"}],"subject":[],"published":{"date-parts":[[2023,3,27]]}}}