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.

中文翻译：

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催化失活、纯化的 RNase H1：一种用于 RNA-DNA 杂交成像的特异性且灵敏的探针

R环是三链核酸结构，在细胞中具有生理和病理作用。R 环成像通常依赖于使用 S9.6 抗体检测这些结构的 RNA-DNA 杂合成分。我们表明，使用这种抗体进行成像可能会存在问题，因为它很容易在体外和体内与双链 RNA (dsRNA) 结合，产生非特异性信号。相比之下，带有 GFP 标记的纯化的、无催化活性的人 RNase H1 (GFP-dRNH1) 是一种用于成像 RNA-DNA 杂交体的更特异的试剂。GFP-dRNH1 与 RNA-DNA 杂交体强烈结合，但不与固定的人类细胞中的 dsRNA 寡核苷酸结合，并且不易结合内源 RNA。此外，我们证明纯化的 GFP-dRNH1 可应用于固定细胞以在诱导后检测杂交体，从而绕过细胞系工程的需要。因此，GFP-dRNH1 有望成为在各种条件下对 RNA-DNA 杂交体进行成像和定量的多功能工具。