Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

• DOI: 10.1083/jcb.202101092
• Publication Date: 2021-07-07T18:55:13Z
• Authors: Magdalena P. Cros...; Joshua R. Brickner; Chenlin Song; Su Mon Thin Zar; Su S. Maw; Frédéric Chédin; Miaw-Sheue Tsai; Karlene A. Cimprich
• Keywords: RNase H; RNA Silencing; Nuclease protection assay

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component these using S9.6 antibody. We show that use this antibody for can be problematic because it readily binds to double-stranded RNA (dsRNA) vitro vivo, giving rise nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged GFP (GFP-dRNH1) is a more specific reagent hybrids. GFP-dRNH1 strongly hybrids but not dsRNA oligonucleotides fixed cells susceptible binding endogenous RNA. Furthermore, we demonstrate purified applied detect after their induction, thereby bypassing need cell line engineering. therefore promises versatile tool quantifying under wide range conditions.