TY - JOUR
T1 - Rational design of novel red-shifted BRET pairs
T2 - Platforms for real-time single-chain protease biosensors
AU - Gammon, Seth T.
AU - Villalobos, Victor M.
AU - Roshal, Mikhail
AU - Samrakandi, Mustapha
AU - Piwnica-Worms, David
PY - 2009/3
Y1 - 2009/3
N2 - 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 (∼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.
AB - 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 (∼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.
KW - Apoptosis
KW - BRET
KW - D-luciferin
KW - Red-shifted
UR - http://www.scopus.com/inward/record.url?scp=66149166193&partnerID=8YFLogxK
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U2 - 10.1002/btpr.144
DO - 10.1002/btpr.144
M3 - Article
C2 - 19330851
AN - SCOPUS:66149166193
SN - 8756-7938
VL - 25
SP - 559
EP - 569
JO - Biotechnology Progress
JF - Biotechnology Progress
IS - 2
ER -