Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Monitoring RNA synthesis is crucial for understanding gene expression and viral replication.
• Existing methods for RNA polymerase activity assays can be complex or lack continuous monitoring capabilities.

Purpose of the Study:

• To develop a convenient and sensitive colorimetric assay for monitoring RNA synthesis by both DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp).
• To enable continuous, real-time detection of polymerase activity using a standard laboratory spectrophotometer.

Main Methods:

• Synthesis of modified nucleotides (PNP-NTPs) with a p-nitrophenyl moiety on the gamma-phosphate.
• Incorporation of PNP-NTPs into RNA by various DdRp and RdRp enzymes.
• Coupling the pyrophosphate release with alkaline phosphatase to generate a detectable colorimetric signal (absorbance at 405 nm).

Main Results:

• The developed assay successfully monitored RNA synthesis by multiple DdRp (T7, SP6) and RdRp (brome mosaic virus, bovine viral diarrhea virus) enzymes.
• A linear increase in absorbance at 405 nm correlated with RNA synthesis, allowing continuous monitoring.
• The nucleotide analogues (PNP-NTPs) demonstrated stability and were utilized by RNA polymerases even in the presence of human plasma.

Conclusions:

• A simple, convenient, and sensitive colorimetric assay for RNA polymerase activity has been established.
• This assay facilitates continuous monitoring of RNA synthesis and is suitable for high-throughput screening.
• The assay is valuable for identifying inhibitors of viral RNA synthesis, aiding in antiviral drug discovery.