title: A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.
abstract: An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan. heejin@pe.res.titech.ac.jp.
authors.forename: Hee-Jin
authors.initials: HJ
authors.lastname: Jeong
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. itayama.0621@pe.res.titech.ac.jp.
authors.forename: Shuya
authors.initials: S
authors.lastname: Itayama
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan. ueda@res.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012235:Rhodamines / Q000737:chemistry; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: C521550:Atto590; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; C005358:tetramethylrhodamine; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus
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