Abstract
The ribonuclease H (RNase H) polymerase-independent cleavage assay allows detection and quantification of RNase H activity of reverse transcriptase (RT) enzymes with a hybrid substrate formed by a fluorescein labeled RNA annealed with Dabcyl DNA (Figure 1). Here we describe a protocol that we have adapted for HIV-1 RT expressed from a p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid and for RT of the prototype foamy virus (PFV RT). Figure 1. Scheme of the principle of the experiment. The RNA substrate (blue) labeled with the fluorophore fluorescein (F, yellow) is annealed with complementary DNA strand (green) labeled with a quencher molecule Dabcyl (D, red). Panel A. In the intact substrate the quencher is so close to the fluorophore that it can quench the fluorescence emitted after excitation. Panel B. After the RNA substrate is cut by the RNase H a few ribonucleotides oligo labeled with the fluorescein is free to escape from the quencher, and to release fluorescence after excitation.
Keywords: RNase H assay, Drug Screening, Ribonuclease activity, RNase H inhibitors , HIV-1 RT RNase H
Materials and Reagents
Equipment
Procedure
Representative data
Figure 2. The results from three independent experiments (biological replicates) are shown as percentage of control. Error bars represent the standard deviation.
Notes
Recipes
Acknowledgments
The p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid was kindly provided by Stuart Le Grice Laboratory (NCI Frederick). The foamy virus reverse transcriptase was kindly provided by Birgitta M. Wöhrl Universität Bayreuth, Lehrstuhl Biopolymere, Bayreuth, Germany. This protocol was modified and adapted from Parniak et al. (2003).
References
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