In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.

Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy.

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids.

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.