A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Nucleic acid polymerases are crucial enzymes involved in DNA and RNA replication and transcription.
• Developing high-throughput screening methods to identify polymerase inhibitors is essential for drug discovery and molecular biology research.

Purpose of the Study:

• To develop a simple, high-throughput screening (HTS)-compatible fluorescence polarization (FP)-based assay for detecting nucleic acid polymerase elongation activity.
• To characterize the utility of this assay for screening compounds that inhibit polymerase binding or elongation.

Main Methods:

• A novel assay utilizing a 5' end-labeled nucleic acid template, termed a polymerase elongation template element (PETE), was designed.
• The assay measures changes in fluorescence polarization signal as the labeled template is drawn into the polymerase active site during elongation.
• The PETE assay was demonstrated in a microtiter plate format using poliovirus RNA-dependent RNA polymerase.

Main Results:

• The FP signal increases upon polymerase-mediated elongation, indicating successful detection of enzyme activity.
• The assay can also detect polymerase binding to the template if the oligonucleotide is sufficiently short.
• The poliovirus RNA-dependent RNA polymerase successfully extended a self-priming hairpin RNA substrate in the PETE assay.

Conclusions:

• The PETE assay is an efficient and HTS-compatible method for monitoring nucleic acid polymerase elongation activity.
• This assay can be used to screen for inhibitors of polymerase binding and elongation activity.
• The developed assay offers a valuable tool for studying polymerase function and discovering novel polymerase-targeting therapeutics.