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A Genetically Encoded Sensor for Real-Time Monitoring of Poly-ADP-Ribosylation Dynamics In Vitro and in Cells.

Alix Thomas¹, Kapil Upadhyaya¹, Daniel Bejan¹

¹Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., L334, Portland, Oregon 97239, United States.

|October 1, 2024

Summary

Researchers developed a new FRET probe (pARS) to monitor ADP-ribosylation dynamics in real-time. This tool allows for sensitive detection of PARylation in live cells and evaluation of PARP inhibitors, advancing DNA damage response studies.

Keywords:

DNA repair FRET fluorescence microscopy posttranslational modifications signal transduction

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Area of Science:

Molecular Biology
Biochemistry
Cell Biology

Background:

ADP-ribosylation is a crucial post-translational modification regulating DNA damage response.
Poly(ADP-ribose) polymerases (PARPs) are key enzymes in this process, with PARP1 being a primary writer.
Real-time monitoring of PARylation dynamics is essential for understanding its regulation in live cells.

Purpose of the Study:

To develop a genetically encoded Förster Resonance Energy Transfer (FRET) probe for monitoring ADP-ribosylation dynamics.
To enable semiquantitative, real-time, and live-cell monitoring of PARylation.
To assess the utility of the probe for studying PARP1 kinetics and evaluating PARP inhibitors.

Main Methods:

Development of a FRET probe (pARS) utilizing a PAR-binding WWE domain flanked by turquoise and Venus fluorescent proteins.
Real-time monitoring of PARP1-dependent PARylation in vitro and in live cells using the pARS probe.
Assessment of pARS probe's sensitivity under varying DNA damage conditions.
In vitro and live-cell evaluation of PARP inhibitor potency using the pARS probe.

Main Results:

The pARS probe provides a ratiometric readout with excellent signal-to-noise for monitoring PARylation.
pARS enables real-time, spatiotemporal tracking of PARP1-mediated PARylation in vitro and in live cells.
The probe demonstrates high sensitivity for detecting PARylation, even under mild DNA damage.
pARS successfully determined the potency of PARP inhibitors both in vitro and in live cells.

Conclusions:

The genetically encoded pARS probe is a robust and user-friendly tool for studying ADP-ribosylation dynamics.
pARS offers valuable insights into PARP1-mediated PARylation kinetics and DNA damage response.
This probe facilitates high-sensitivity detection of PARylation and evaluation of PARP inhibitors in live-cell settings.