Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids

Haibo Yang; Li Lan

doi:10.1007/978-1-0716-2477-7_17

Immunostaining and Protein Pull-Down of Methyl-5-Cytosine-Marked RNA:DNA Hybrids

Methods Mol Biol. 2022:2528:271-276. doi: 10.1007/978-1-0716-2477-7_17.

Authors

Haibo Yang^{1

2}, Li Lan^{3

4}

Affiliations

¹ Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA.
² Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
³ Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA. llan1@mgh.harvard.edu.
⁴ Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. llan1@mgh.harvard.edu.

PMID: 35704197
DOI: 10.1007/978-1-0716-2477-7_17

Abstract

RNA:DNA hybrids not only function in various physiological cellular processes but also represent a threat to genome integrity. The methyl-5-cytosine (m⁵C) marked RNA:DNA hybrids containing the m⁵C modified RNA strand add another layer of regulation to the R-loop structure. Here we describe the detection of m⁵C modifications in the context of RNA:DNA hybrids using immunostaining, and identification of specific binding partners of m⁵C marked RNA:DNA hybrids using pull-down method.

Keywords: Biotin-pull down; Immunostaining; R-loop; RNA:DNA hybrid; m5C.

MeSH terms

Cytosine*
DNA / genetics
RNA* / genetics

Substances

RNA
Cytosine
DNA