Ultrasensitive evaluation of Ribonuclease H activity using a DNAzyme-powered on-particle DNA walker

Abstract

The evaluation of Ribonuclease H (RNase H) activity is very important for anti-HIV drug development and the study of cellular processes, including DNA replication, DNA repair and transcription. However, the highly sensitive detection of RNase H activity with a simple strategy remains challenging. Here, a DNAzyme-powered on-particle DNA walker was proposed as a signal amplifier to evaluate the RNase H activity at levels as low as 0.00847 U mL−1. Pre-caged enzyme strands and substrate strands of Mn2+-specific DNAzyme were modified at the interface of gold nanoparticles, forming the DNA walker probes. In the absence of RNase H, a minimum fluorescent signal was observed due to excellent ability of gold nanoparticles to quench the fluorophore that labeled the substrate strand. In the presence of RNase H, the DNA/RNA hybrid complex was hydrolyzed to release single stranded DNA, which was able to hybridize to the locker strand that caged the enzyme strand. Following the addition of Mn2+, the active forms of DNAzymes were cleaved at the RNA site of substrate strands, releasing enhanced fluorescent molecules. Finally, the walker strands traveled over the AuNPs like a machine to amplify the signal, thus indirectly measuring the activity of RNase H. This approach proved to be an ultrasensitive and simple method for the evaluation of RNase H activity in cell extracts and has also been successfully used to evaluate the effects of inhibitors.