RNase H Hydrolysis Assay of Modified RNA

Recombinant human RNase H1 in pBAD-His plasmid was expressed in BL21 Escherichia coli and purified by affinity chromatography followed by gel permeation. The catalytically active RNase H domain fragment of HIV-1 RT was expressed from plasmid pCSR231 (a generous gift from Dr. Daria Hazuda, Merck, West Point, PA) and purified as previously described.^{39 (link)} RNA template was 5′-radiolabeled with γ-P³²-ATP (PerkinElmer) using T4 polynucleotide kinase and annealed with either DNA (B1), 2-F-araU (B2), or 2′,4′-diF-araU (B3) modified substrate. RNase H hydrolysis reactions were conducted at room temperature in 50 mM Tris-HCl, pH 8.0, and 50 mM KCl buffer with 20 nM each duplex and 100 nM enzyme. Reaction was started by adding 5 mM MgCl₂ and quenched at different time points by 95% formamide and 10 mM EDTA, pH 8.0, with a trace amount of bromophenol blue dye. Products of reactions were separated using 20% denaturing PAGE and analyzed by phosphorimaging.