Bright bioluminescent BRET sensor proteins for measuring intracellular caspase activity

A. den Hamer, P. Dierickx, R. Arts, J.S.P.M. de Vries, L. Brunsveld, M. Merkx

Research output: Contribution to journalArticleAcademicpeer-review

26 Citations (Scopus)
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Abstract

FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.

Original languageEnglish
Pages (from-to)729-734
Number of pages6
JournalACS Sensors
Volume2
Issue number6
DOIs
Publication statusPublished - 23 Jun 2017

Keywords

  • BRET
  • caspase sensor
  • mNeonGreen
  • NanoLuc
  • plate reader assay
  • single-cell imaging

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