http://www.bio-protocol.org/e1561 Vol 5, Iss 16, Aug 20, 2015 RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes Angela Corona and Enzo Tramontano * Department of Life and Environmental Sciences, University of Cagliari, Monserrato, Italy * For correspondence: tramon@unica.it [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. Materials and Reagents 1. Reverse transcriptase enzyme (HIV-1 RT or PFV RT). HIV-1 RT expressed and purified from p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid (Corona et al., 2014a), PFV RT provided by Birgitta M. Wöhrl from Universität Bayreuth, Lehrstuhl Biopolymere, Bayreuth, Germany (Corona et al., 2014b). 2. Distilled water (DNase/RNase-free UltraPure TM ) (Life Technologies, Invitrogen TM , catalog number: 10977-015) 3. 5'-GAUCUGAGCCUGGGAGCU-FLUORESCEIN-3' (HPLC, dry, QC: Mass Check) (Metabion) 4. 5'-DABCYL-AGCTCCCAGGCTCAGATC-3' (HPLC, dry, QC: Mass Check) (Metabion) Copyright © 2015 The Authors; exclusive licensee Bio-protocol LLC. 1 Please cite this article as: Angela and Enzo, (2015). RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes, Bio-protocol 5 (16): e1561. DOI: 10.21769/BioProtoc.1561.