Anti-DNA-RNA Hybrid Antibody, clone S9.6

DNA-RNA hybrids are a natural occurrence within eukaryotic cells and their level are high at sites of high transcriptional activity. They are non-canonical nucleic acid structures with transcriptional regulatory functions. Their presence is reported to predispose a locus to chromosomal breakage. A locus forming an DNA:RNA creates a double-stranded A/B intermediate conformation, with a second target for single-stranded nucleic acid binding proteins on the complementary, displaced DNA strand. They are shown to be resistant to the activity of DNA methyltransferases. The formation of DNA:RNA hybrids has been associated with a number of neurological diseases. Mutations in the DNA:RNA helicase senataxin (SETX) are implicated in the dominant juvenile form of amyotrophic lateral sclerosis type 4 and a recessive form of ataxia oculomotor apraxia type 2.

Clone S9.6 bound the DNA-RNA heteropolymer and poly(I)-poly(dC) equally, but 100-fold higher levels of poly(A)-poly(dT) were required to achieve a similar degree of binding. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by clone S9.6 (Boguslawski, S.J., et al. (1986). J. Immunol Methods. 89(1):123-130).

Target DNA-RNA heteroduplex (R loop) structure is not sequence- or species-specific.

DNA-RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase (Boguslawski, S.J., et al. (1986). J. Immunol Methods. 89(1):123-130).

Anti-DNA-RNA Hybrid, clone S9.6, Cat. No. MABE1095, is a highly specific mouse monoclonal antibody, that targets DNA-RNA hybrid and has been tested in Affinity Binding Assay, Chromatin Immunoprecipitation (ChIP), ChIP-seq, Dot Blot, Immunocytochemistry, and Immunoprecipitation.

Dot Blot Analysis: 0.2 µg/mL from a representative lot detected an enhanced level of DNA-RNA hybrids in genomic extracts from RNase H-deficient yeast strain than extracts from wild-type strain (Courtesy of Lorenzo Costantino, Ph.D., Koshland Lab, University of California at Berkley, U.S.A.).

Affinity Binding Assay: Clone S9.6 bound the DNA-RNA heteropolymer and poly(I)-poly(dC) equally, but 100-fold higher levels of poly(A)-poly(dT) were required to achieve a similar degree of binding. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by clone S9.6 (Boguslawski, S.J., et al. (1986). J. Immunol Methods. 89(1):123-130).

Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot detected increased DNA RNA hybrids at four actively transcribed genes upon shRNA-mediated knockdown of BRCA1 or BRCA2, but not PCID2 or RAD51 in HeLa cells (Bhatia, V., et al. (2014). Nature. 511(7509):362-365).

Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot detected R-loops formed over beta-actin gene using HeLa chromatin preparation. RNase H treatment of the chromatin preparation prevented clone S9.6 from immunoprecipitating target chromatin fragments (Skourti-Stathaki, K., et al. (2011). Mol. Cell. 42(6):794-805).

Chromatin Immunoprecipitation-sequencing (ChIP-seq) Analysis: A representative lot detected genome-wide distribution of DNA-RNA hybrids in budding yeast by ChIP-seq analysis (El Hage, A., et al. (2014). PLoS Genet. 10(10):e1004716).

Immunocytochemistry Analysis: Representative lots immunolocalized nuclear R loops by fluorescent immunocytochemistry staining of methanol-fixed H1 human embryonic stem cells (hESCs) and formaldehyde-fixed HeLa cells (Bhatia, V., et al. (2014). Nature. 511(7509):362-365; Ginno, P.A., et al. (2012). Mol. Cell. 45(6):814-825).

Immunoprecipitation Analysis: A representative lot immunoprecipitated in vitro transcribed R-loop substrate (DNA-RNA hybrid), but not doouble-stranded DNA (dsDNA) (Ginno, P.A., et al. (2012). Mol. Cell. 45(6):814-825).

Research Category
Epigenetics & Nuclear Function

Evaluated by Immunocytochemistry in HeLa cells.

Immunocytochemistry Analysis: A 1:50 dilution of this antibody immunolocalized nuclear and mitochondrial DNA-RNA hybrids in 4% paraformaldehyde-fixed, 0.3% Triton X-100-permeabilized HeLa cells.

Format: Purified

Protein G purified.

Purified mouse IgG2aκ in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Stable for 1 year at 2-8°C from date of receipt.

Concentration: Please refer to lot specific datasheet.

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