title: Rational design of novel red-shifted BRET pairs: Platforms for real-time single-chain protease biosensors.
abstract: Bioluminescence resonance energy transfer (BRET) systems to date have been dominated by use of blue-green Renilla luciferase (Rluc) as the energy donor. Although effective in many cases, the expense and unfavorable biochemical attributes of the substrate (phenylcoelenterazine) limit utility of Rluc-based BRET systems. Herein we report a series of novel BRET pairs based on luciferases that utilize D-luciferin, resulting in red-shifted photonic outputs, favorable biochemical attributes, and increased efficacy. We developed a modified Förster equation to predict optimal BRET luciferase donor-fluorophore pairs and identified tdTomato as the optimal red fluorophore acceptor for click beetle green luciferase (CBG). A prototypical single-chain protease biosensor, capable of reporting on executioner caspase activity in live cells and in real-time, was generated by inserting a DEVD linker between CBG and tdTomato and validated in vitro with recombinant caspases and in cellulo with apoptosis-sensitive and -resistant cell lines. High signal-to-noise ratios ( approximately 33) and Z' factors (0.85) were observed in live cell longitudinal studies, sufficient for high-throughput screening. Thus, we illustrate a general methodology for the rational design of new BRET systems and provide a novel single-chain BRET protease biosensor that is long lived, red-shifted, and utilizes D-luciferin.
authors.affiliation: Dept. of Molecular Biology and Pharmacology, and Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Seth T
authors.initials: ST
authors.lastname: Gammon
authors.forename: Victor M
authors.initials: VM
authors.lastname: Villalobos
authors.forename: Mikhail
authors.initials: M
authors.lastname: Roshal
authors.forename: Mustapha
authors.initials: M
authors.lastname: Samrakandi
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms