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 Km and kcat 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.

2D Boron Sheets: Structure, Growth, and Electronic and Thermal Transport Properties

“…Taking Cu(111) surface as a representative, Liu, Gao and Zhao explored various boron sheet and boron clusters on Cu(111) surfaces. [133] They found the infinite boron sheets are stable on Cu(111) surface, which is also confirmed by successive simulation. [118] Most important of all, the planar B N (N = 1-20) cluster is also the ground-state structure on Cu(111) surfaces (Figure 23a), in comparison with those ring configurations.…”

“…Liu et al revealed that the hexagonal holes can arise at the edge of boron domain through edge reconstruction. [133] The formation barrier for a hole is only 0.53 eV, which is easy to happen even under room temperature. After arising along the edge, the hexagonal can also diffuse into interior region easily.…”

“…Ag (111) surface is the key to stabilize the flat silicene clusters, ensuring the 2D growth of silicene. [133] Besides, the diffusion barrier of silicon atoms on Ag(111) surface is very low, providing enough diffusion under a low growth temperature. In contrast, too strong interaction between silicene and Rh (111) surface may break up the honeycomb structure of silicene.…”

Target‐Activated DNA Polymerase Activity for Sensitive RNase H Activity Assay

“…However, the signal response in these methods is not large enough for the sensitive determination of RNase H activity, which is attributed to the fact that a single target only promotes a single signaling event. In addition, these methods often require the direct modification of detection probes with fluorophore and quencher, which can interfere with the effective RNase H‐catalyzed reactions, consequently raising issues such as poor sensitivity and reliability of the assays …”

“…In addition, these methods often require the direct modification of detection probes with fluorophore and quencher, which can interfere with the effective RNase H-catalyzed reactions, consequently raising issues such as poor sensitivity and reliability of the assays. [13,25,26] In an attempt to address the issues in previous fluorometric methods, we developed a novel method for sensitive RNase H activity assays by designing a unique detection probe composed of a DNA polymerase-specific aptamer (DPA) and an RNA-containing strand (RS). The proposed method relies on our recent finding that a DNA aptamer, containing 5′ target-specific overhang sequence, can regulate the DNA polymerase activity in response to the target molecule.…”