Highly sensitive detection of RNase H via a novel DNA/RNA heteroduplex combining isothermal exponential amplification strategy

Ribonuclease H (RNase H) plays a crucial role in a variety of cellular processes and is emerging as an essential therapeutic target for many diseases. Various methods have been constructed to assay RNase H activity, but these methods are either time-consuming or have poor sensitivity. In this study, we developed a novel strategy that combined a specially designed DNA/RNA chimeric substrate with exponential amplification reaction (EXPAR) for rapid and sensitive detection of RNase H activity. In the presence of RNase H, the RNA strand of the DNA/RNA heteroduplex was specifically degraded, forming a 3′-hydroxyl group in the locking primer for recognition and extension by DNA polymerase. This ultimately triggered EXPAR and produced a large number of single-stranded DNA, which was monitored in real-time with fluorescent dye. Under optimized conditions, the proposed strategy can detect as low as 6 × 10⁻¹⁰ U/μL of RNase H, which was at least 1000 times more sensitive than several reported methods. Furthermore, we demonstrated the usage of this method for RNase H inhibitor analysis and practical application in complex biological samples, including serum and tumor cell extracts. Therefore, these results suggested that the developed method is a promising tool for highly sensitive detection of RNase H and RNase H-associated disease diagnosis.