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A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

Jeong HJ, Itayama S, Ueda H - Biosensors (Basel) (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

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Related in: MedlinePlus

Fluorescence image of (a) ATTO520-; (b) tetramethylrhodamine (TAMRA)-; and (c) ATTO590-incorporated Q’-bodies. Scheme above each gel is the chemical structure of the dye used.
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biosensors-05-00131-f003: Fluorescence image of (a) ATTO520-; (b) tetramethylrhodamine (TAMRA)-; and (c) ATTO590-incorporated Q’-bodies. Scheme above each gel is the chemical structure of the dye used.

Mentions: Twelve kinds of Q’-bodies with different configurations and fluorophores were synthesized and purified by immobilized metal affinity chromatography, and the fluorescence images of the SDS-PAGE gels were taken (Figure 3). Clear bands of ATTO520- and TAMRA- labeled Q’-bodies were observed at above the 25 kD marker, indicating successful expression of these proteins with incorporated fluorophores. On the other hand, in the case of purified ATTO590-labeled Q’-bodies with two dyes, faint bands of full-length proteins were observed. When non-purified proteins were applied to the gel, a prominent band at smaller molecular weight of ~15 kD was observed in the cases of K*R* and R*K*. In this study, we used aminoacylated amber suppressor tRNAs to introduce unnatural amino acids [25]. Since the amber codon is a stop codon, the synthesis of double-labeled full-length proteins would be less efficient due to imperfect suppression efficiency. As a result, the full-length protein was mostly removed after purification via the C-terminal His6-tag. In particular, the larger size of ATTO590 than other two (molecular weights of ATTO590, TAMRA and ATTO520 are 691.2, 430.5 and 466.9, respectively) and its linear and consecutive five benzene rings might be unfavorable for the efficient incorporation. Nevertheless, the purified proteins with single ATTO590 dye were mostly full-length.


A signal-on fluorosensor based on quench-release principle for sensitive detection of antibiotic rapamycin.

Jeong HJ, Itayama S, Ueda H - Biosensors (Basel) (2015)

Fluorescence image of (a) ATTO520-; (b) tetramethylrhodamine (TAMRA)-; and (c) ATTO590-incorporated Q’-bodies. Scheme above each gel is the chemical structure of the dye used.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493541&req=5

biosensors-05-00131-f003: Fluorescence image of (a) ATTO520-; (b) tetramethylrhodamine (TAMRA)-; and (c) ATTO590-incorporated Q’-bodies. Scheme above each gel is the chemical structure of the dye used.
Mentions: Twelve kinds of Q’-bodies with different configurations and fluorophores were synthesized and purified by immobilized metal affinity chromatography, and the fluorescence images of the SDS-PAGE gels were taken (Figure 3). Clear bands of ATTO520- and TAMRA- labeled Q’-bodies were observed at above the 25 kD marker, indicating successful expression of these proteins with incorporated fluorophores. On the other hand, in the case of purified ATTO590-labeled Q’-bodies with two dyes, faint bands of full-length proteins were observed. When non-purified proteins were applied to the gel, a prominent band at smaller molecular weight of ~15 kD was observed in the cases of K*R* and R*K*. In this study, we used aminoacylated amber suppressor tRNAs to introduce unnatural amino acids [25]. Since the amber codon is a stop codon, the synthesis of double-labeled full-length proteins would be less efficient due to imperfect suppression efficiency. As a result, the full-length protein was mostly removed after purification via the C-terminal His6-tag. In particular, the larger size of ATTO590 than other two (molecular weights of ATTO590, TAMRA and ATTO520 are 691.2, 430.5 and 466.9, respectively) and its linear and consecutive five benzene rings might be unfavorable for the efficient incorporation. Nevertheless, the purified proteins with single ATTO590 dye were mostly full-length.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus