Victor Villalobos MD, PhD

Publication Details

  • Rational Design of Novel Red-Shifted BRET Pairs: Platforms for Real-Time Single-Chain Protease Biosensors BIOTECHNOLOGY PROGRESS Gammon, S. T., Villalobos, V. A., Roshal, M., Samrakandi, M., Piwnica-Worms, D. 2009; 25 (2): 559-569

    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.

    View details for DOI 10.1021/bp.144

    View details for Web of Science ID 000265572700033

    View details for PubMedID 19330851

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