Abstract
An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase-hexahistidine-2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.
Highlights
An R loop is a special triple-stranded nucleic acid structure formed when nascent RNA invades double-stranded DNA during transcription, resulting in a DNA-RNA hybrid and a displaced single- stranded DNA
In an attempt to overcome the limits of the S9.6 antibody and catalytically inactive full-length RNase H1, we took advantage of the DNA-RNA hybrid binding proprieties of the N-terminal hybrid-binding domain (HBD) domain of RNase H1 [22], which contains a three-stranded antiparallel sheet and two short helices (Fig. 1A)
For specific DNA-RNA hybrid recognition in vitro, we designed two glutathione S-transferase (GST)- and His6-tagged sensor proteins (GST-His6-HBD and GST-His6-2×HBD) with tandem repeats of HBD separated by a flexible 5×Glycine linker (Fig. 1A)
Summary
An R loop is a special triple-stranded nucleic acid structure formed when nascent RNA invades double-stranded DNA (dsDNA) during transcription, resulting in a DNA-RNA hybrid and a displaced single- stranded DNA (ssDNA). Several genome-wide R loop mapping methods were developed, using either the S9.6 monoclonal antibody (mAb) or catalytically inactive ribonuclease (RNase) H1 for specific DNA-RNA hybrid (a defining feature of R loops) binding and capturing [11]. The predominant strategy for genome-wide profiling of R loops is the DNA-RNA immunoprecipitation sequencing (DRIP-seq), which captures DNA-containing R loop fragments using the S9.6 antibody before sequencing [12,13,14,15,16,17]. The specificity of the S9.6 antibody has been questioned recently for accurate quantification and mapping of genuine R loops [11, 19, 20]. The digestion efficiency and bias in chromatin fragmentation by restriction enzymes could potentially compromise R loop mapping resolution in DRIP-related approaches [21]
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