Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging.
Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA-DNA hybrid imaging.
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DOI：
10.1083/jcb.202101092
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发表时间：
2021-09-06
期刊：
影响因子：
--
通讯作者：
Cimprich KA
中科院分区：
文献类型：
--
作者：
Crossley MP;Brickner JR;Song C;Zar SMT;Maw SS;Chédin F;Tsai MS;Cimprich KA
Crossley等人证明gfp标记的催化失活的RNase H1蛋白是细胞r环成像的通用工具。他们还表明，它明显比常用的S9.6抗体更具特异性，后者检测到相当多的非特异性信号。r环是一种三链核酸结构，在细胞中具有生理和病理双重作用。r环成像通常依赖于使用S9.6抗体检测这些结构的RNA-DNA杂交成分。我们发现，使用这种抗体进行成像可能会有问题，因为它在体外和体内都很容易与双链RNA （dsRNA）结合，产生非特异性信号。相比之下，用GFP标记的纯化的、无催化活性的人RNase H1 （GFP- drnh1）是一种更特异的RNA-DNA杂交成像试剂。在固定的人类细胞中，GFP-dRNH1与RNA - dna杂交体结合强烈，但不与dsRNA寡核苷酸结合，并且不容易与内源性RNA结合。此外，我们证明纯化的GFP-dRNH1可以应用于固定细胞，在诱导后检测杂交，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成像和定量RNA-DNA杂交的通用工具。
Crossley et al. demonstrate that GFP-tagged, catalytically inactive RNase H1 protein is a versatile tool for imaging cellular R-loops. They also show that it is significantly more specific than the commonly used S9.6 antibody, which detects considerable nonspecific signal. 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.
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DOI：
10.1126/science.aan6490
发表时间：
2018-01-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Kabeche L;Nguyen HD;Buisson R;Zou L
通讯作者：
Zou L
影响因子：
11.4
作者：
Nowotny, Marcin;Cerritelli, Susana M.;Yang, Wei
通讯作者：
Yang, Wei
影响因子：
16
作者：
Nowotny, Marcin;Gaidamakov, Sergei A.;Yang, Wei
通讯作者：
Yang, Wei
影响因子：
16
作者：
Hatchi, Elodie;Skourti-Stathaki, Konstantina;Ventz, Steffen;Pinello, Luca;Yen, Angela;Kamieniarz-Gdula, Kinga;Dimitrov, Stoil;Pathania, Shailja;McKinney, Kristine M.;Eaton, Matthew L.;Kellis, Manolis;Hill, Sarah J.;Parmigiani, Giovanni;Proudfoot, Nicholas J.;Livingston, David M.
通讯作者：
Livingston, David M.
影响因子：
16
作者：
Chappidi, Nagaraja;Nascakova, Zuzana;Janscak, Pavel
通讯作者：
Janscak, Pavel