RNase A activity analysis and imaging using label-free DNA-templated silver nanoclusters

Area of Science:

• Biochemistry
• Nanotechnology
• Analytical Chemistry

Background:

• Ribonuclease A (RNase A) plays crucial roles in various biological processes.
• Accurate detection and quantification of RNase A are essential for biological studies and disease diagnostics.
• Existing methods for RNase A detection often lack sensitivity or require complex procedures.

Purpose of the Study:

• To develop a label-free, ultra-sensitive, and turn-on fluorescence method for RNase A detection.
• To utilize enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as a fluorescence probe.
• To enable real-time monitoring of RNase A activity in vitro and in living cells.

Main Methods:

• Design of a system where an RNA strand hybridizes with a DNA template, quenching the fluorescence of DNA-AgNCs.
• Utilizing the RNA/DNA duplex as a substrate for RNase A.
• Monitoring the restoration of fluorescence signal upon RNase A-mediated degradation of the RNA strand.

Main Results:

• A positive linear relationship was observed between fluorescence signal and RNase A concentration (0.2 pg/μL to 10 pg/μL).
• Achieved a low detection limit of 0.098 pg/μL for RNase A.
• Successfully applied the method for potential inhibitor screening, kinetic studies, and monitoring dynamic changes of RNase A in living cells.

Conclusions:

• The developed method provides a simple, sensitive, and turn-on approach for RNase A assay.
• The strategy holds promise for applications in enzyme activity studies, drug screening, and in vivo monitoring.
• This enhanced DNA-AgNCs system offers a versatile platform for sensitive biomolecule detection.