TY  - JOUR
TI  - RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes
AU  - Corona, Angela 
AU  - Tramontano, Enzo 
VL  - 5
IS  - 16
PY  - 2015
DA  - 2015/08/20
SP  - e1561
C1  - Bio-protocol 2015;5:e1561
DO  - 10.21769/BioProtoc.1561
UR  - https://doi.org/10.21769/BioProtoc.1561
AB  - 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.
KW  - RNase H assay
KW  - Drug Screening
KW  - Ribonuclease activity
KW  - RNase H inhibitors
KW  - HIV-1 RT RNase H
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False