title: A biocompatible in vivo ligation reaction and its application for noninvasive bioluminescent imaging of protease activity in living mice.
abstract: The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH(2)-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.
authors: Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology of Lausanne, LCBIM, CH-1015 Lausanne, Switzerland.
authors: Aurélien
authors: A
authors: Godinat
authors: Hyo Min
authors: HM
authors: Park
authors: Stephen C
authors: SC
authors: Miller
authors: Ke
authors: K
authors: Cheng
authors: Douglas
authors: D
authors: Hanahan
authors: Laura E
authors: LE
authors: Sanman
authors: Matthew
authors: M
authors: Bogyo
authors: Allen
authors: A
authors: Yu
authors: Gennady F
authors: GF
authors: Nikitin
authors: Andreas
authors: A
authors: Stahl
authors: Elena A
authors: EA
authors: Dubikovskaya
mesh_terms: D000818:Animals; D017209:Apoptosis; D052160:Benzothiazoles / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry; D005260:Female; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D049408:Luminescent Agents / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D009570:Nitriles / Q000737:chemistry*; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
chemical_list: C578355:2-cyanobenzothiazole; D052160:Benzothiazoles; C532924:D-luciferin; D049408:Luminescent Agents; D009570:Nitriles; D009842:Oligopeptides; C511444:aspartyl-glutamyl-valyl-aspartyl-aminoluciferin; D049454:Firefly Luciferin; D049409:Luciferases, Firefly; D010447:Peptide Hydrolases; D013917:Thrombin; D053148:Caspase 3; D053179:Caspase 7; D003545:Cysteine