Laboratory of Chemical Biology and State Key laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, PR China.
Chemistry. 2010 Feb 22;16(8):2605-10. doi: 10.1002/chem.200902166.
We demonstrate a unique quadruplex-based fluorescence assay for sensitive, facile, real-time, and label-free detection of RNase H activity and inhibition by using a G-quadruplex formation strategy. In our approach, a RNA-DNA substrate was prepared, with the DNA strand designed as a quadruplex-forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G-rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G-quadruplex binder, N-methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high-throughput screening of enzyme inhibitors and demonstrates that the structure of the G-quadruplex can be used as a functional tool in specific fields in the future.
我们展示了一种独特的基于四链体的荧光分析方法，用于灵敏、简便、实时和无标记检测 RNase H 的活性和抑制作用，该方法基于 G-四链体形成策略。在我们的方法中，制备了 RNA-DNA 底物，其中 DNA 链设计为形成四链体的寡聚物。在单离子存在下，当 RNA 链被 RNase H 切割时，释放出的富含 G 的 DNA 链折叠成四链体，并与特定的 G-四链体结合物 N-甲基 mesoporphyrin IX（NMM）相互作用；这导致荧光显著增加，并作为反应的报告。该新型测定方法设计简单，操作快速，比其他方法更方便，更有前景。整个过程不到 30 分钟，检测限比之前的报道要好很多，至少与之相当。不需要对 RNA 或 DNA 进行复杂的实验技术或化学修饰。该测定可以使用普通分光光度计完成，并且避免了可能对催化剂动力学行为的干扰。我们的方法为酶抑制剂的高通量筛选提供了一个理想的系统，并表明 G-四链体的结构将来可以在特定领域用作功能工具。
