Abstract
ANNEXIN V belongs to a family of phospholipid binding proteins which is able to bind to negatively charged phospholipids such as phosphatidylserine (PS) in the presence of a high affinity Ca2+ ion. When apoptosis occurs, even at early stage, PS will be exposed to the outside of the cell surface from the cytoplasm side of membrane leaflets., Therefore ANNEXIN V has been suggested as a bio-marker for imaging early apoptotic events of various cell death including those in disease conditions. However, most ANNEXIN V-based apoptotic detecting techniques were in vitro approaches. Here, we presented a new BRET (Bioluminescence Resonance Energy Transfer) based genetic coded biosensor by fusing ANNEXIN V and a BRET version of NanoLuc (teLuc) for both in vitro and in vivo apoptosis detection. The BRET feature of this new sensor makes it convenient to be applied to both conventional fluorescent-based in vitro apoptosis detection and bioluminescence-based animal live imaging for in vivo study. Because of its robust bioluminescence signal, it is possible to perform the evaluation of the disease-induced apoptotic damage and recovery process directly at deep tissue level in live animal.
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Acknowledgements
We thank Dr. Xiaoxuan Xu for technique support when conducting live imaging. We thank Mr. Zhikang Zhao for helping animal care and maintenance. This work was supported by the National Key Basic Research Program (NKBRP) (BK20130057) from Ministry of Science and Technology of the People's Republic of China and the National Natural Science Foundation of China. (Grant # 3224003212).
Funding
National Key Basic Research Program (NKBRP) (BK20130057) from Ministry of Science and Technology of the People's Republic of China and the National Natural Science Foundation of China. (Grant # 3224003212).
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10495_2021_1693_MOESM1_ESM.tif
BRET property of AnVPb and AnV-Antares2 probes. Bioluminescence emission signal of lysis supernatants from 9 individual clones for each probe was collected after mixed with substrate DTZ (30 µM). Averaged bioluminescence curves of AnVPb (a) and AnV-Antares2 (b) demonstrated a much higher activity (>100 times) in AnVPb supernatant. Individual bioluminescence curves of AnVPb clones (c) presented an identical emission curve peaked at 516 nm. While the BRET curves of AnV-Antares2 clones varied a lot, indicating a less stability of AnV-Antares2 (tif 3249 kb)
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Zhang, Y., Hu, J., Yu, M. et al. A novel BRET based genetic coded biosensor for apoptosis detection at deep tissue level in live animal. Apoptosis 26, 628–638 (2021). https://doi.org/10.1007/s10495-021-01693-x
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DOI: https://doi.org/10.1007/s10495-021-01693-x