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

Crossley, Magdalena P.; Brickner, Joshua R.; Song, Chenlin; Zar, Su Thin; Maw, Su S.; Chédin, Frédéric; Tsai, Miaw-Sheue; Cimprich, Karlene A.

doi:10.1083/jcb.202101092

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

Journal Article · Wed Jul 07 04:00:00 EDT 2021 · Journal of Cell Biology

DOI:https://doi.org/10.1083/jcb.202101092· OSTI ID:2470862

Crossley, Magdalena P. ^[1]; ^[1]; Song, Chenlin ^[1]; Zar, Su Thin ^[2]; Maw, Su S. ^[2]; ^[3]; ^[2]; ^[1]

Stanford University, CA (United States). School of Medicine
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
University of California, Davis, CA (United States)

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

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; National Institutes of Health (NIH); Leukemia & Lymphoma Society (LLS); Jane Coffin Childs Fund (JCC); V Foundation for Cancer Research

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470862

Journal Information:: Journal of Cell Biology, Journal Name: Journal of Cell Biology Journal Issue: 9 Vol. 220; ISSN 0021-9525

Publisher:: Rockefeller University PressCopyright Statement

Country of Publication:: United States

Language:: English

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