A dual surface plasmon resonance assay for the determination of ribonuclease H activity.
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.
Sípová
Hana
H
Institute of Photonics and Electronics, Academy of Sciences of the Czech Republic, Chaberská 57, 182 51 Prague, Czech Republic.
Vaisocherová
Hana
H
Stěpánek
Josef
J
Homola
Jiří
J
D015342:DNA Probes; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes; D015347:RNA Probes; D016914:Ribonuclease H / Q000032:analysis* / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D013696:Temperature
D015342:DNA Probes; D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D015347:RNA Probes; D016914:Ribonuclease H