Published: January 22nd, 2021
This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.
The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.
This protocol provides a step-by-step guide to a dot-blot assay that allows a quick comparative assessment of the abundance of R-loop, a three-stranded nucleic acid structure. R-loop forms when RNA invades a double-stranded DNA to generate an RNA-DNA hybrid and displaces the other DNA strand. R-loops are found in different stages of the lifecycle of RNA. In the transcriptional complex, the nascent RNA is synthesized complementary to the template DNA, and the non-template strand is displaced. The short RNA-DNA hybrid (<10 bp) is resolved to free the nascent RNA so it can leave the RNA polymerase complex through the exit channel1,2. Outside of the transcriptional complex, the nascent RNA is close to its DNA template, which is still slightly unwound from being copied, thus the RNA can rehybridize with its template DNA forming R-loops3. Additionally, R-loops can form when replication and transcription complexes collide4, and in antisense transcription5. Given the many opportunities for their formation, R-loops are not rare, and can be found in 3-5% of the human genome6, depending on the cell's transcription status. R-loops are found in gene promoters7 and termination5 sites in mRNA, and along ribosomal RNA8 as well as transfer RNA9. R-loops are also in telomeric regions of chromosomes.
R-loops play a regulatory role. They regulate gene expression by affecting transcription at promoters10,11, mediating class-switch recombination12, and facilitating CRISPR-based genome editing13,14,15. Like many cellular events, R-loop abundance is tightly titrated; too many or too few R-loops impact normal cell function16,17. R-loops are regulated by a variety of proteins including RNase H, senataxin, and other helicases that unwind the RNA-DNA hybrids18,19,20,21,22.
To monitor the abundance of R-loops, genome-wide methods first enrich for R-loops with the antibody S9.68,23,24 or with other nucleases25 including RNase H10,26,27, and then assess the number of enriched R-loops by sequencing. Early versions of these sequencing-based methods did not achieve adequate sequence coverage to allow precise quantitation, but rapid improvement in sequencing technologies now allows locus-by-locus R-loop analysis. Immunofluorescence techniques have also been used to quantify and localize R-loops10,17. These methods are comprehensive, but they are not practical in many clinical settings or as initial assessments since they require expensive equipment and specialized analysis.
A procedure that can be done uniformly across laboratories in clinical settings is needed. Dot-blots provide such an option since they can be carried out without any specific equipment or computational analysis. As a preliminary step or in clinical settings to evaluate the effects of mutations on R-loops, these dot-blots must provide sensitive and specific results. Here, we describe our assay that identifies R-loops specifically; it excludes signals from double-stranded (ds) DNA, double-stranded RNA, and single-stranded RNA. Our protocol uses the S9.6 antibody27 to identify RNA-DNA hybrids in R-loops and incorporates RNase H, an endoribonuclease that cleaves and therefore leads to the degradation of the RNA in an RNA-DNA hybrid20,28, to ensure that the detected signals are those of hybrids. We also incorporated RNase T1 that cleaves single-stranded RNA at guanine29,30, and RNase III that cleaves double-stranded RNA including stem-loops31,32 to check for nonspecific signals. The S9.6 antibody recognizes RNA-DNA hybrids of varying lengths, even those that are only 8 nucleotides long33.
Here, we present the protocol that begins with nucleic acid isolation followed by dot-blot preparation, and R-loop detection with S9.6 antibody. Our protocol includes steps to ensure that equal amounts of samples are loaded, and the signals are specific. It provides oligonucleotides to serve as positive and negative controls. This is a quick, easy, and user-friendly method to assess R-loop abundance.
1. Cell lysis for nuclear fractionation
2. Purification of genomic DNA (which includes RNA-DNA hybrids)
3. Blotting DNA samples (which include RNA-DNA hybrids) onto nylon membranes
4. RNA-DNA hybrid detection with S9.6 antibody
5. Ribonuclease treatments to evaluate signal specificity
NOTE: RNase treatment should be performed on the nucleic acid samples to demonstrate the specificity of S9.6 binding. Treatment with RNase H, but not RNase T1 or RNase III should result in a reduction in S9.6 immunostaining.
6. Preparation of oligonucleotide controls to evaluate signal specificity
NOTE: Oligonucleotide controls can be used to demonstrate the specificity of S9.6 binding. S9.6 recognizes RNA-DNA hybrids, but not dsDNA or dsRNA controls, as has been previously reported34.
7. Quantification and normalization of S9.6 R-loop signal intensity using ImageJ.
Enzymatic treatment to evaluate the specificity of S9.6 (RNA-DNA) antibody.
Primary human skin fibroblasts were grown17. DNA with RNA-DNA hybrids was isolated and quantified. Two µg of the samples were digested with RNase T1, RNase H, or RNase III for 15 min at 37 °C. A mock sample was also analyzed for comparison to the RNase-treated samples. Samples (200, 100, 50, 25, 12.5, or 6.25 ng) were blotted onto two different membranes as described in section 3. The membranes were crosslinked, blocked and one of them was probed with S9.6 antibody (Figure 1A).
The results showed that the S9.6 signal correlates with the abundance of the loaded sample. Treatment with RNase H, but not RNase T1 or RNase III results in a reduction in S9.6 staining.
A second membrane was probed with a dsDNA antibody (Figure 1B) for the normalization. Image J was used to analyze the signal intensities. The 50 ng samples were selected for quantification as the signal intensities from the S9.6 and dsDNA antibodies were within the dynamic range. Signal intensities were normalized to those in mock samples. Data are shown in Figure 2.
S9.6 antibody dot-blot using synthetic nucleotide controls.
To evaluate the specificity of the S9.6 antibody, we used oligonucleotides corresponding to dsRNA, dsDNA, and RNA-DNA as described in section 6. A dilution series of RNA-DNA, dsRNA, and dsDNA nucleotides were prepared and blotted onto the nylon membrane as described in section 3. The membrane was probed with the S9.6 antibody (Figure 3). Results showed that the S9.6 antibody binds specifically to RNA-DNA hybrids in a dose-dependent manner and showed minimal cross-reactivity to dsRNAs and dsDNAs.
Figure 1: Specificity of S9.6 as shown by dot-blot loaded with nucleic acids from human fibroblasts. Nucleic acid samples from human fibroblasts were either mock treated or treated with RNase T1, RNase H, or RNase III and then loaded onto nylon membranes in a dilution series of 200, 100, 50, 25, 12.5, and 6.25 ng per 2 µL dot. Membranes were then probed with S9.6 antibody (A), or dsDNA antibody (B). Please click here to view a larger version of this figure.
Figure 2: Quantification of S9.6 R-loop staining. 50 ng samples from Figure 1 were selected for quantification with ImageJ. S9.6 signal was divided by dsDNA signal intensity, then normalized to the mock sample following the steps outlined in section 7. Please click here to view a larger version of this figure.
Figure 3: S9.6 dot-blot using oligonucleotide controls. S9.6 antibody dot-blot against a dilution series of synthetic oligonucleotides as dsRNA, dsDNA, or RNA-DNA hybrid. S9.6 binds specifically to RNA-DNA hybrids in a dose-dependent manner. Please click here to view a larger version of this figure.
|Cell lysis buffer
|0.5M PIPES (pH 8.0)
|Nuclear lysis buffer
|1M Tris-HCl (pH 8.0)
|1M Tris-Cl, pH 8.5
Table 1. Preparation of buffers
|Verify cell fractionation
|The quality of nuclear and cytoplasmic separation can be evaluated by adding standard protease inhibitor cocktails to the cell and nuclear lysis buffers (Table 1). The cytoplasmic and nuclear fractions can be evaluated by western blotting analysis to confirm adequate restriction of labeling with cytoplasmic markers (for example, GAPDH or HSP90) in cytoplasmic fractions and labeling with nuclear markers (for example, HDAC1 or Histone H3) in nuclear fractions. The contribution of mitochondrial contamination in the nuclear fraction can be evaluated by qPCR analysis with probes specific for mitochondrial DNA.
|Sample is too viscous
|Cell number is too high.
|Reduce the DNA by half and continue with the sonication step 2.1
|No visible pellet
|Insufficient starting material or loss during extraction.
|Start from the beginning using more cells.
|Low DNA concentration
|Not enough DNA for dilutions
|Sample won't saturate into the membrane
|Soak the membrane in 1x TBST. Allow excess buffer to dry and start at step 3.4
|Patchy or speckled pattern is detected
|Add 0.1% Sodium dodecyl sulfate (SDS) to the sample and continue at step 3.1
|The dots have a "coffee ring" appearance
|Add 0.01% Sarkosyl to the sample and continue at step 3.1
|The RNaseH control shows no decrease in signal
|The ribonuclease digestion is incomplete.
|Increase incubation or increase enzyme concentration.
|No signal for the oligo controls
|Duplex wasn't formed. Oligonucleotides weren't properly annealed.
|Verify the ratios of oligonucleotides and annealing buffer.
|S9.6 signal isn't specific to hybrids
|S9.6 antibody batch has non-specific binding
|Validate the sensitivity and specificity of new batches of S9.6 antibody with the use of either RNase enzyme treatments or synthetic oligonucleotide analysis.
Table 2: Troubleshooting
|ssRNA, top strand
|ssDNA, top strand
|ssRNA, bottom strand
|ssDNA, bottom strand
Table 3. Control oligonucleotide sequences
The 3-stranded nucleic acids, R-loops, form in different stages during the lifecycle of RNA and are increasingly found to regulate cellular processes. To fully understand R-loops, reliable techniques for R-loop detection are necessary. Here, we describe an approach to interrogate the abundance of R-loops using S9.6 antibody8,23,24. This method allows for a quick assessment of R-loop abundance from cells and tissue-culture samples. It does not require special equipment, or a large quantity of starting material. It ensures specific and reproducible results using a combination of RNase treatments.
Some have reported concerns about the specificity of the S9.6 antibody. As with any reagent, there may be batch to batch variability with the S9.6 antibody. Our protocol includes RNase H, RNase T1 and RNase III to check signal specificity. In addition, we use synthetic oligonucleotides to ensure the specificity of each batch of S9.6 antibody.
R-loop biology is a growing field; the development of reliable detection and quantification methods, such as the one presented here, will facilitate mechanistic studies to elucidate when R-loops form, how they are regulated, and what they regulate. With appropriate controls, this dot-blot assay is a simple method to screen for R-loop abundance in clinical and research settings.
The authors have nothing to disclose.
This work was supported by the Howard Hughes Medical Institute and Intramural Research at the National Institute of Neurological Disorders and Stroke.
We thank Dr. Stephen Leppla for providing batches of S9.6 antibody for analysis. We also thank Dr. Dongjun Li for his assistance with ribonuclease treatments.
|Anti-RNA-DNA hybrid antibody (S9.6)
|Hybond N+ nylon membrane
|GE healthcare Life Sciences
|NP-40 (Igepal CA-630)
|PIPES (0.5M, pH 8.0)
|New England Biolabs
|sodium acetate (3M, pH 5.2)
|Tris-buffered saline (10X)
|Tris-HCl (1M, pH 8.0)
|UV Stratalinker 2400
|Whatman marking pen
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