title: A novel luminescent biosensor for rapid monitoring of IP3 by split-luciferase complementary assay.
abstract: Inositol 1,4,5-trisphosphate (IP(3)) is a crucial second messenger that regulates complicated signaling processes in various physiological events. Alteration in its content has been observed in many diseases. Hence, development of a high-throughput screening system to monitor temporal changes of IP(3) is essential for screening of new potential therapeutic compounds. Toward a simple, sensitive and rapid method for measuring IP(3), we describe the development and application of a novel biosensor based on luciferase fragment assisted complementation strategy, which converts the ligand-induced conformational changes to light. Designed sensor comprising the IP(3)-binding core domain of IP(3)-receptor fused between complementary non-functional fragments of firefly luciferase allows direct detection of IP(3) in presence of luciferin substrate both in cell lysate and in living cells. According to the result presented in this manuscript, the screening time was very fast and maximum response was obtained up to 11-fold higher than untreated cells. Moreover, the designed biosensor was able to monitor release of IP(3) upon induction by different inducers like Bradykinin and ATP. The current biosensor not only provides a specific IP(3) detector in vitro but also facilitates monitoring of the response of IP(3) in living organisms.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Farangis
authors.initials: F
authors.lastname: Ataei
authors.forename: Masoud
authors.initials: M
authors.lastname: Torkzadeh-Mahani
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry*; D008156:Luciferases / Q000032:analysis / Q000737:chemistry*; D008163:Luminescent Measurements / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate; D008156:Luciferases