College of Biology, Hunan University, Changsha, 410082, China.
People's Hospital of Hunan Province, Changsha, 410082, China.
Mikrochim Acta. 2019 May 7;186(6):335. doi: 10.1007/s00604-019-3425-6.
A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg. The recycle cleavage can directly result in the amplified signal emitted by the FAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5 U·mL. The detection limit of 0.018 U·mL is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screened for their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection. Graphical abstract The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.
一种新的荧光法用于检测 RNase H 活性，该方法将 DNAzyme 与还原氧化石墨烯 (rGO) 结合使用。在没有 RNase H 的情况下，由于 FAM 标记探针强烈吸附在 rGO 上，其荧光被猝灭。当存在 RNase H 时，它可以从 DNA-RNA 嵌合链上释放出活性 DNAzyme。在辅助因子 Mg 的帮助下，该 DNAzyme 可以触发 rA 位点处信号探针的切割。这种循环切割可以直接导致由 FAM 标记的短片段发出放大信号。该方法允许在 0.01 至 5 U·mL 的线性范围内检测 RNase H 的活性。通过空白信号三倍标准差的原理计算得出，该方法的检测限为 0.018 U·mL。然后，筛选了针对 RNase H 的天然化合物以研究其抑制作用。在所研究的化合物中，有 5 种以浓度依赖的方式被筛选为 RNase H 抑制剂，有 4 种被鉴定为激活剂。最后，该方法可靠地用于区分人血清中 RNase H 活性的差异。结果发现，丙型肝炎病毒感染患者的 RNase H 活性升高。
