Real time monitoring of junction ribonuclease activity of RNase H using chimeric molecular beacons

Abstract

As a highly conserved damage repair protein, except for hydrolysis of DNA–RNA heteroduplex endonucleolytically, RNase H can cleave RNA–DNA junctions in Okazaki fragment processing through its junction ribonuclease (JRNase) activity. We report here a real time fluorescence method for detecting JRNase activity of RNase H with high accuracy by applying chimeric molecular beacons as substrates. The detection limit of E. coli RNase H is 0.5 U ml⁻¹. The K_m and k_cat are 20 nM and 0.6 s⁻¹, respectively. In addition, we used the method to investigate the effect of chemical drugs on the enzyme and found that both penicillin and streptomycin sulfate inhibit its activity with the IC₅₀ values of 0.2 and 0.07 mM, respectively. Finally, we applied the method to reliably detect the JRNase level in tumor cells. In summary, these data indicate that the simple, rapid and sensitive method can be hopefully applied for high-throughput detection of samples and drug screening in vitro.