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FAR-RED FRET-SENSOR FOR DETERMINATION OF CASPASE-3 ACTIVITY

Goryashchenko A.S, Zherdeva V.V, Scherbo D.S, Shemiakina I.I, Savitsky A.P
Apoptosis is one of the variants of the programmed cell death. The main effector enzyme in the process of apoptosis is the caspase-3. Level of caspase-3 activity is a good marker for the detection of apoptosis. Real-time determination of the caspase-3 activity in living cells became possible by means of genetically encoded FRET-sensors. Use of far-red fluorescent proteins minimizes the level of background fluorescence and increases the depth of penetration of a signal. The purpose of this work was to characterize the genetically encoded caspase-3 FRET-sensor FusionRed-Linker1-eqFP670 containing the far-red fluorescent proteins FusionRed and eqFP670. The hydrodynamic radius of a sensor was determined as 6,2 nm as a result of dynamic light scattering experiments, that in spherical model approach corresponds to the molecular weight of 244 kDa and the tetrameric structure in solution. Decrease of fluorescence intensity of the sensor in comparison with individual FusionRed protein testifies the presence of energy transfer between the donor and acceptor. We also calculated FRET efficiency in the sensor and found that it is about 24%. Cleavage of our sensor by caspase-3 in vitro was proved by fluorescence intensity measurements and by SDS-PAGE.

Библиографическая ссылка

Горященко А.С, Жердева В.В, Щербо Д.С, Шемякина И.И, Савицкий А.П ДАЛЬНЕКРАСНЫЙ FRET-СЕНСОР ДЛЯ ДЕТЕКЦИИ АКТИВАЦИИ КАСПАЗЫ-3 // Научное обозрение. Химические науки. 2020. № 1. С. 11-11;
URL: https://science-chemistry.ru/en/article/view?id=5 (дата обращения: 27.07.2026).