Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location

Author(s)

Gabriel, Gabriele V. M. ^{[1, 2, 3]} ; Viviani, Vadim R. ^{[1, 2, 3]}

Affiliation

^[1] Fed Univ Sao Carlos UFSCar, Grad Sch Evolut Genet & Mol Biol, Sao Carlos, SP - Brazil

^[2] Fed Univ Sao Carlos UFScar, Grad Sch Biotechnol & Environm Monitoring, Sorocaba, SP - Brazil

^[3] Fed Univ Sao Carlos UFSCar, Dept Phys Chem & Math, Lab Biochem & Biotechnol Bioluminescence, Sorocaba, SP - Brazil

• Document type: Journal article
• Source: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES; v. 13, n. 12, p. 1661-1670, 2014.
• Web of Science Citations: 9

Abstract

Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes. (AU)

• FAPESP's process: 11/23961-0 - pH-sensitive luciferases: structural origin of pH-sensitivity, and suitability of use in cell biosensors for pH and heavy metals
• Grantee: Vadim Viviani
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/04477-9 - Developing of bioluminescent ratiometric biosensors for pH and divalent metals based on the engineering of the pH-sensing moiety of firefly luciferases
• Grantee: Gabriele Verônica de Mello Gabriel
• Support Opportunities: Scholarships in Brazil - Doctorate