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Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

Ekaterina O Serebrovskaya1, Nadezda M Podvalnaya1,2, Varvara V Dudenkova3

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Scientists developed a new fluorescent sensor, sPARroW, to track poly-(ADP-ribose) (PAR) levels in real-time within living cells. This tool helps study cellular processes like DNA repair and cell death.

Keywords:
DNA damage resposeFRETPARWWE-domaifluorescent proteinsensor

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Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Poly-(ADP-ribosyl)-ation (PARylation) is a crucial post-translational modification involved in DNA repair, replication, transcription, and cell death.
  • Monitoring PAR levels in live cells is essential for understanding these fundamental cellular processes.

Purpose of the Study:

  • To design and validate a genetically encoded fluorescent sensor for visualizing poly-(ADP-ribose) (PAR) dynamics in live mammalian cells.
  • To establish a tool for real-time monitoring of PAR accumulation and depletion in response to cellular stress.

Main Methods:

  • Development of a Förster resonance energy transfer (FRET)-based biosensor, sPARroW.
  • Utilized the WWE domain of RNF146 as a PAR-binding module.
  • Fused fluorescent proteins (Turquoise2 and Venus) to the WWE domain for FRET detection.
  • Tested the sensor's efficacy in live mammalian cells under various stress conditions.

Main Results:

  • The sPARroW sensor successfully detected and visualized PAR accumulation and depletion in response to hydrogen peroxide, UV irradiation, and hyperthermia.
  • Demonstrated the sensor's ability to monitor dynamic changes in PAR levels in real-time.
  • Validated the specificity of the WWE domain for targeting PAR.

Conclusions:

  • The sPARroW sensor provides a novel and effective method for studying PARylation in live cells.
  • This tool facilitates research into the roles of PARylation in cellular responses to DNA damage and other stresses.
  • Enables deeper insights into the mechanisms of DNA repair, transcription, and cell death pathways.