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Related Experiment Videos

A fluorescence-based assay for multisubunit DNA-dependent RNA polymerases.

Peter Kuhlman1, Heidi L Duff, Ashley Galant

  • 1Department of Chemistry and Biochemistry, Denison University, Granville, OH 43023, USA. kuhlman@denison.edu

Analytical Biochemistry
|December 24, 2003
PubMed
Summary

This study introduces a new, cost-effective RNA polymerase activity assay using RiboGreen fluorescence. This method replaces expensive radioisotopes, enabling high-throughput drug screening and analysis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • DNA-dependent RNA polymerases are crucial enzymes studied since the 1960s.
  • Interest persists in RNA polymerase function, cellular roles, and drug targeting.
  • Traditional assays use radioisotopes, which are expensive and have short shelf lives.

Purpose of the Study:

  • To develop a novel, cost-effective assay for measuring RNA polymerase activity.
  • To replace radioisotope-based methods with a fluorescence-based detection system.
  • To facilitate high-throughput screening for drug discovery and biochemical analysis.

Main Methods:

  • Utilized the RiboGreen dye for sensitive fluorescence detection of RNA transcripts.
  • Developed a microplate-based assay format for parallel sample processing.

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  • Incorporated DNase digestion and ultrafiltration to remove template DNA and enable solvent exchange.

    • Main Results:

    • The RiboGreen assay provides a rapid and cost-effective alternative to radioisotope methods.
    • The assay is suitable for processing numerous samples in parallel, ideal for screening.
    • The method allows for accurate transcription level comparisons under varying solvent conditions.

    Conclusions:

    • The developed RiboGreen fluorescence assay is a viable, efficient tool for studying RNA polymerase activity.
    • This assay is well-suited for drug screening and analyzing enzymatic reaction products.
    • It offers significant advantages in cost, speed, and handling compared to traditional methods.