Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe

Area of Science:

• Biochemistry and Molecular Biology
• Analytical Chemistry
• Biomarker Discovery

Background:

• Ribonuclease A (RNase A) is crucial in enzymology and implicated in disease development, making it a potential theranostic biomarker.
• Existing methods for RNase A detection may lack sensitivity, cost-effectiveness, or applicability for screening and diagnostics.

Purpose of the Study:

• To develop a simple, sensitive, and low-cost assay for Ribonuclease A (RNase A) detection.
• To utilize the assay for screening natural compounds as RNase A modulators.
• To investigate the impact of drug treatments and Hepatitis B virus (HBV) infection on RNase A activity.

Main Methods:

• Development of a fluorogenic substrate assay combined with reduced graphene oxide (rGO) for enhanced RNase A detection.
• Application of the assay for high-throughput screening of natural compounds targeting RNase A.
• Analysis of RNase A activity in response to drug treatments (G-10, Chikusetsusaponin V) and HBV infection in biological samples.

Main Results:

• The assay achieved a low detection limit of 0.05 ng/mL for RNase A.
• Fourteen natural compounds were identified as RNase A activators; G-10 and Chikusetsusaponin V upregulated RNase A in tumor cells.
• RNase A activity was significantly inhibited in HBV infected groups, and heavy metal ions (Ba2+, Co2+, Pb2+, As3+, Cu2+) showed dose-dependent inhibition.

Conclusions:

• The developed rGO-based assay is rapid, sensitive, and practical for RNase A quantification in biological samples.
• The assay facilitates effective screening of natural compounds for RNase A modulation in vitro and in vivo.
• RNase A levels are altered by HBV infection and heavy metal exposure, suggesting its role in disease pathogenesis and toxicology.