A scintillation proximity assay for poly(ADP-ribose) polymerase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Poly(ADP-ribose) polymerase (PARP) is a crucial nuclear enzyme involved in DNA repair.
• PARP activation by DNA damage leads to the synthesis of poly(ADP-ribose) from NAD.
• Current radioactive assays for PARP are hindered by a rate-limiting separation step, impeding large-scale screening for inhibitors.

Purpose of the Study:

• To develop a novel, efficient assay for Poly(ADP-ribose) polymerase (PARP) activity.
• To facilitate high-throughput screening for small-molecule PARP inhibitors.
• To overcome limitations of conventional radioactive PARP enzyme assays.

Main Methods:

• Development of a scintillation proximity assay (SPA) utilizing biotinylated NAD.
• Demonstration of PARP incorporating biotinylated ADP-ribose units into poly(ADP-ribose) polymer.
• Adaptation of the PARP-SPA assay to a 96-well format for automated screening.

Main Results:

• The developed PARP-SPA assay directly measures PARP activity without a separation step.
• Biotinylated NAD allows the synthesized polymer to bind and excite streptavidin-conjugated scintillation beads.
• The assay is suitable for high-throughput screening, enabling efficient identification of PARP inhibitors.

Conclusions:

• The novel PARP-SPA assay offers a streamlined and efficient method for detecting PARP activity.
• This assay is readily adaptable for automated high-throughput screening of potential PARP inhibitors.
• The developed method significantly improves upon conventional assays, facilitating drug discovery efforts.