A quadruplex-based, label-free, and real-time fluorescence assay for RNase H activity and inhibition.
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.
Hu
Dan
D
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.
Pu
Fang
F
Huang
Zhenzhen
Z
Ren
Jinsong
J
Qu
Xiaogang
X
D001483:Base Sequence; D001681:Biological Assay; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D054856:G-Quadruplexes; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D016914:Ribonuclease H / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D016615:Telomere / Q000737:chemistry; D013997:Time Factors
D005456:Fluorescent Dyes; D004247:DNA; D016914:Ribonuclease H