Abstract
We developed a new versatile strategy that allows the detection of several classes of RNases (i.e., targeting ss- or ds-RNA, DNA/RNA hetero-hybrid or junctions) with higher sensitivity than existing assays. Our two-step approach consists of a DNA-RNA-DNA chimeric Hairpin Probe (cHP) conjugated to magnetic microparticles and containing a DNAzyme sequence in its terminal region, and molecular beacons for fluorescence signal generation. In the first step, the digestion of the RNA portion of the cHP sequences in presence of RNases leads to the release of multiple copies of the DNAzyme in solution. Then, after magnetic washing, each DNAzyme molecule elicits the catalytic cleavage of numerous molecular beacons, providing a strong amplification of the overall sensitivity of the assay. We successfully applied our approach to detect very low concentrations of RNase A, E. coli RNase I, and RNase H. Furthermore, we analyzed the effect of two antibiotics (penicillin and streptomycin) on RNase H activity, demonstrating the applicability of our strategy for the screening of inhibitors. Finally, we exploited our system to detect RNase activity directly in crude biological samples (i.e., blood and saliva) and in cell culture medium, highlighting its suitability as cheap and sensitive tool for the detection of RNase levels.
Highlights
We developed a new versatile strategy that allows the detection of several classes of RNases with higher sensitivity than existing assays
Our two-step approach consists of a DNA-RNA-DNA chimeric Hairpin Probe conjugated to magnetic microparticles and containing a DNAzyme sequence in its terminal region, and molecular beacons for fluorescence signal generation
After magnetic washing, each DNAzyme molecule elicits the catalytic cleavage of numerous molecular beacons, providing a strong amplification of the overall sensitivity of the assay
Summary
We developed a new versatile strategy that allows the detection of several classes of RNases (i.e., targeting ssor ds-RNA, DNA/RNA hetero-hybrid or junctions) with higher sensitivity than existing assays. Experimental data indicate that our hybrid assay allows to evaluate the effects of inhibitors with high sensitivity, representing an interesting tool for testing and screening of anti-viral drugs. We have shown an innovative and versatile strategy for the detection of RNases activity, based on a hybrid chimeric probe, allowing the highly sensitive analysis of different classes of RNases, such as RNase A, RNase I and the junction activity of RNase H These characteristics, coupled with the small volume of samples needed by our approach, make our assay suitable for future implementations, such as for low-cost Lab-On-a-Chip and high-throughput screening applications
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