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.
Highlights
Rapamycin (Sirolimus) is an antibiotic discovered from Streptomyces hygroscopicus in the soil of Easter island [1], and it is routinely used as a primary immunosuppressor for preventing kidney transplant rejection
(FKBP12-rapamycin binding), and the formation of FKBP12-rapamycin-FRB ternary complex inhibits an mTOR, a Ser/Thr protein kinase that modulates signaling pathways regulating cellular growth and division [4,5,6]
An FKBP12-FRB gene with N-terminal (G3S)2 and internal (G4S)3 linkers was prepared by splice overlap extension (SOE) PCR as follows
Summary
Rapamycin (Sirolimus) is an antibiotic discovered from Streptomyces hygroscopicus in the soil of Easter island [1], and it is routinely used as a primary immunosuppressor for preventing kidney transplant rejection. Variant [16] and firefly luciferase (Fluc) [17,18] are reported All these methods use two proteins as a pair of probes comprised of two independent binding domains FKBP12 and FRB to probe rapamycin. When antigen binds to antibody, the quenching of fluorophore is released by the antigen-induced structural change of Fv. when antigen binds to antibody, the quenching of fluorophore is released by the antigen-induced structural change of Fv Based on this principle, simple mixing of a Q-body with an antigen and subsequent fluorescence intensity measurement enables antigen quantitation in a simple and convenient manner. Simple mixing of a Q-body with an antigen and subsequent fluorescence intensity measurement enables antigen quantitation in a simple and convenient manner It has a great potential for biosensing applications requiring no additional reagents and washing steps. We constructed several fluorophore-protein conjugates named Q’-bodies, mimicking the Q-body concept, using an interacting protein pair FKBP12 and FRB whose interaction is mediated by rapamycin (Figure 1)
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