A dual surface plasmon resonance assay for the determination of ribonuclease H activity

作者：Sipova Hana; Vaisocherova Hana; Stepanek Josef; Homola Jiri^*

来源：Biosensors and Bioelectronics, 2010, 26(4): 1605-1611.

DOI：10.1016/j.bios.2010.08.011

• Biosensor
• Surface plasmon resonance
• Antisense
• Ribonuclease H
• Enzyme activity assay

There is a demand for efficient tools for the monitoring of RNase H activity. We report on a new assay which allows for simultaneous (1) real-time monitoring of RNase H activity and (2) detection of cleavage reaction products. The dual assay is implemented using a multichannel surface plasmon resonance (SPR) biosensor with two independently functionalized sensing areas in a single fluidic path. In the first sensing area the RNA cleavage by RNase H is monitored, while the products of the cleavage reaction are captured in the second sensing area with specific DNA probes. The assay was optimized with respect to AON concentration and temperature. A significant improvement was obtained with special chimeric probes, which contain RNA substrate for RNase H and a longer deoxyribonucleotide tail, which enhances the SPR signal. It has been shown that RNase H stabilizes the RNA:DNA hybrid duplex before the cleavage. The potential of the assay is demonstrated in the study in which the ability of natural and modified oligonucleotides to activate RNase H is examined.

• 出版日期2010-12-15