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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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Schematic representation of Q’-body genes. Asterisk denotes an amber codon.
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biosensors-05-00131-f002: Schematic representation of Q’-body genes. Asterisk denotes an amber codon.

Mentions: We constructed rapamycin Q’-body by tethering the FKBP12 and FRB with a flexible (G4S)3 linker, and incorporated fluorophore(s) to the specific site(s) of the fusion protein (Figure 1). We named this fluorescent protein as a “Q’-body” due to similarity in concept to Q-body, which is made of a single chain antibody (the two variable region fragments VH and VL of antibody tethered by a flexible linker) with site-specifically incorporated fluorophore(s). Since the position and number of the fluorophore might affect the fluorescent response, we constructed four different types of Q’-body genes, *KR, *K*R, *RK, and *R*K (Figure 2), wherein *KR is an FKBP12-FRB type gene with a 5'-terminal ProX-tag, which has an amber codon to introduce a dye (MSKQIEVNY*SNET, asterisk denotes an amber codon), and a 3'-terminal His-tag. The sequence of *K*R is the same as *KR, except for an additional amber codon introduced in the middle of the interdomain linker. Considering the fact that more Trp residues exist in FRB (four) than FKBP12 (one), constructs with reversed domain order *RK and *R*K were made similarly. To investigate the effect of dyes, we used three rhodamine derivatives to examine their performance as a suitable label for Q’-body. In addition to TAMRA and ATTO520, which are proven good labels for a Q-body, a new fluorophore ATTO590 with longer excitation/emission wavelengths with possible in vivo applications was examined.


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

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

Schematic representation of Q’-body genes. Asterisk denotes an amber codon.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00131-f002: Schematic representation of Q’-body genes. Asterisk denotes an amber codon.
Mentions: We constructed rapamycin Q’-body by tethering the FKBP12 and FRB with a flexible (G4S)3 linker, and incorporated fluorophore(s) to the specific site(s) of the fusion protein (Figure 1). We named this fluorescent protein as a “Q’-body” due to similarity in concept to Q-body, which is made of a single chain antibody (the two variable region fragments VH and VL of antibody tethered by a flexible linker) with site-specifically incorporated fluorophore(s). Since the position and number of the fluorophore might affect the fluorescent response, we constructed four different types of Q’-body genes, *KR, *K*R, *RK, and *R*K (Figure 2), wherein *KR is an FKBP12-FRB type gene with a 5'-terminal ProX-tag, which has an amber codon to introduce a dye (MSKQIEVNY*SNET, asterisk denotes an amber codon), and a 3'-terminal His-tag. The sequence of *K*R is the same as *KR, except for an additional amber codon introduced in the middle of the interdomain linker. Considering the fact that more Trp residues exist in FRB (four) than FKBP12 (one), constructs with reversed domain order *RK and *R*K were made similarly. To investigate the effect of dyes, we used three rhodamine derivatives to examine their performance as a suitable label for Q’-body. In addition to TAMRA and ATTO520, which are proven good labels for a Q-body, a new fluorophore ATTO590 with longer excitation/emission wavelengths with possible in vivo applications was examined.

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