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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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(a) Comparison of the fluorescence responses of Q’-body upon addition of 1 μM rapamycin. Error bars represent ±1 SD (n = 3). (b) Fluorescence time-course of ATTO520-incorporated *RK type Q’-body after adding rapamycin. (c) Fluorescence spectra of ATTO520-incorporated *RK type Q’-body in the presence of rapamycin at indicated concentrations. (d) The same as in (c) except that DMSO, the solvent of rapamycin, was added. (e) Rapamycin concentration-dependent fluorescence increase of ATTO520-incorporated *RK type Q’-body Error bars represent ±1 SD (n = 20).
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biosensors-05-00131-f004: (a) Comparison of the fluorescence responses of Q’-body upon addition of 1 μM rapamycin. Error bars represent ±1 SD (n = 3). (b) Fluorescence time-course of ATTO520-incorporated *RK type Q’-body after adding rapamycin. (c) Fluorescence spectra of ATTO520-incorporated *RK type Q’-body in the presence of rapamycin at indicated concentrations. (d) The same as in (c) except that DMSO, the solvent of rapamycin, was added. (e) Rapamycin concentration-dependent fluorescence increase of ATTO520-incorporated *RK type Q’-body Error bars represent ±1 SD (n = 20).

Mentions: We measured the fluorescence intensity of purified Q’-body in the absence and presence of 1 µM rapamycin (Figure 4a). As a result, the fluorescence intensity was increased within 2 minutes after adding rapamycin (Figure 4b). However, the obtained responses were different from each other depending on the configuration of Q’-body and the dye used. Among the four constructs, *RK-type Q’-bodies showed the highest rapamycin-dependent fluorescence enhancement, regardless of the fluorophores. Since there are four Trp residues in FRB and one in FKBP12, it is probable that the fluorophore has more chances to be quenched when it is placed closer to FRB. Unexpectedly, unlike our previous Q-bodies [21], double-labeled Q’-bodies showed lower responses than the single-labeled ones. Among the three dyes used, ATTO520-labeled Q’-bodies showed the highest response for all the types of constructs. This difference most likely reflects that of fluorophore’s chemical structures. ATTO520 is less hydrophobic and smaller in size, and has a positive charge unlike TAMRA and ATTO590, which might result in its lower tendency to dimer-induced quenching (H-dimer formation) than TAMRA and ATTO590. Possibly, the interface between FKBP12 and FRB is less hydrophobic than that of VH and VL, thus the less hydrophobic dye was more prone to enter the interface and more efficiently quenched. Also, in the cases of TAMRA and ATTO590, intermolecular dye-dye interaction might have made these dyes quenched irrespective of rapamycin, thus might have prevented their efficient responses. Alternatively, as the molecular size of ATTO520 is smaller than other dyes and the distance between the dye and protein would be shorter, the dye might be more effectively quenched by Trp residues [26].


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

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

(a) Comparison of the fluorescence responses of Q’-body upon addition of 1 μM rapamycin. Error bars represent ±1 SD (n = 3). (b) Fluorescence time-course of ATTO520-incorporated *RK type Q’-body after adding rapamycin. (c) Fluorescence spectra of ATTO520-incorporated *RK type Q’-body in the presence of rapamycin at indicated concentrations. (d) The same as in (c) except that DMSO, the solvent of rapamycin, was added. (e) Rapamycin concentration-dependent fluorescence increase of ATTO520-incorporated *RK type Q’-body Error bars represent ±1 SD (n = 20).
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00131-f004: (a) Comparison of the fluorescence responses of Q’-body upon addition of 1 μM rapamycin. Error bars represent ±1 SD (n = 3). (b) Fluorescence time-course of ATTO520-incorporated *RK type Q’-body after adding rapamycin. (c) Fluorescence spectra of ATTO520-incorporated *RK type Q’-body in the presence of rapamycin at indicated concentrations. (d) The same as in (c) except that DMSO, the solvent of rapamycin, was added. (e) Rapamycin concentration-dependent fluorescence increase of ATTO520-incorporated *RK type Q’-body Error bars represent ±1 SD (n = 20).
Mentions: We measured the fluorescence intensity of purified Q’-body in the absence and presence of 1 µM rapamycin (Figure 4a). As a result, the fluorescence intensity was increased within 2 minutes after adding rapamycin (Figure 4b). However, the obtained responses were different from each other depending on the configuration of Q’-body and the dye used. Among the four constructs, *RK-type Q’-bodies showed the highest rapamycin-dependent fluorescence enhancement, regardless of the fluorophores. Since there are four Trp residues in FRB and one in FKBP12, it is probable that the fluorophore has more chances to be quenched when it is placed closer to FRB. Unexpectedly, unlike our previous Q-bodies [21], double-labeled Q’-bodies showed lower responses than the single-labeled ones. Among the three dyes used, ATTO520-labeled Q’-bodies showed the highest response for all the types of constructs. This difference most likely reflects that of fluorophore’s chemical structures. ATTO520 is less hydrophobic and smaller in size, and has a positive charge unlike TAMRA and ATTO590, which might result in its lower tendency to dimer-induced quenching (H-dimer formation) than TAMRA and ATTO590. Possibly, the interface between FKBP12 and FRB is less hydrophobic than that of VH and VL, thus the less hydrophobic dye was more prone to enter the interface and more efficiently quenched. Also, in the cases of TAMRA and ATTO590, intermolecular dye-dye interaction might have made these dyes quenched irrespective of rapamycin, thus might have prevented their efficient responses. Alternatively, as the molecular size of ATTO520 is smaller than other dyes and the distance between the dye and protein would be shorter, the dye might be more effectively quenched by Trp residues [26].

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