Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids

BMC Research Notes

Michael R Lieber¹  Kefei Yu²  Chih-Lin Hsieh¹  Nicholas R Pannunzio¹  Zheng Z Zhang¹ 

[1] Departments of Pathology, Biochemistry & Molecular Biology; Molecular Microbiology & Immunology; Urology, University of Southern California Keck School of Medicine, 1441 Eastlake Ave., Rm. 5428, Los Angeles 90089-9176, CA, USA;Department of Microbiology and Molecular Genetics, Michigan State University, 5175 Biomedical Physical Sciences, East Lansing 48824, MI, USA

关键词: Nucleic acid structure;    S9.6 monoclonal antibody;    Immunoprecipitation;    R-loop;    Immunogloblulin heavy chain class switch recombination;   

Others  :  1164300
DOI  :  10.1186/s13104-015-1092-1

 received in 2014-10-21, accepted in 2015-03-24,  发布年份 2015

【 摘 要 】

Background

Long genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a K_D that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions.Here we investigate whether RNase A is needed to obtain reliable IP with S9.6.

Findings

As our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop.

Conclusion

Any use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by forming RNA:RNA regions or short, transient RNA:DNA duplexes. Caution should be used when interpreting S9.6 data, and confirmation by independent structural and functional methods is essential.

【 授权许可】

   
2015 Zhang et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

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