Fluorescence Assay for Ribonuclease H Based on Nonlabeled Substrate and DNAzyme Assisted Cascade Amplification

As a highly conserved damage repair protein, RNase H can specifically hydrolyze RNA in DNA–RNA chimeric strands. DNAzyme, a synthetic single-stranded DNA consisting of binding and catalytic sites, can cleave RNA in the presence of cofactors. In this study, we establish a highly sensitive RNase H assay assisted with DNAzyme’s cleavage property. A DNA–RNA chimeric strand, which contains DNAzyme sequences, is used as the hydrolysis substrate of RNase H. The RNase H hydrolysis of the chimeric substrate results in the release of DNAzyme. Subsegment DNAzyme digest, a molecular beacon, causes a “turn-on” fluorescence signal by disrupting its hairpin structure. Furthermore, the fluorescence signal is amplified by cyclic digestion of DNAzyme to the substrate of molecular beacon. Under the optimal conditions, the detection limit of RNase H is 0.01 U/mL, which is superior to those of several alternative approaches. Additionally, the method was further used for RNase H detection in heterogeneous biological samples as well as to investigate the effects of natural compounds on this enzyme. In summary, these results show that the method not only provides a universal platform for monitoring RNase H activity but also shows great potential in biomedical studies and drug screening.