DNA-RNA immunoprecipitation (DRIP) is used to detect and analyze RNA-DNA hybrids, particularly R-loops, within genomic DNA. This protocol, adapted from Professor Frédéric Chédin’s lab, outlines a detailed workflow for preparing R-loop samples and performing immunoprecipitation using the S9.6 antibody. The method enables researchers to study transcriptional dynamics and genome stability by mapping hybrid regions using qPCR, microarrays, or sequencing.

Importantly, DRIP enables genome-wide mapping of RNA-DNA hybrids, allowing comprehensive analysis of R-loop distribution across the genome.

Introduction

DRIP is an antibody-based technique designed to isolate and study RNA-DNA hybrids formed during transcription. These hybrids, known as R-loops, play critical roles in gene regulation and genome integrity. R-loops frequently form at gene promoters and within the gene body, often associated with transcriptional pause sites and regulated by RNA Polymerase II. The protocol uses the S9.6 monoclonal antibody to selectively bind RNA-DNA hybrids, enabling their enrichment and downstream analysis. This method is used in molecular biology and epigenetics to investigate transcriptional pausing, replication stress, and chromatin structure.

Background and principles

The DRIP technique is based on the principle of immunoprecipitation, where specific antibodies are used to capture RNA-DNA hybrids from genomic DNA. R-loops are formed when nascent RNA hybridizes with the DNA template strand, displacing the non-template strand. Template DNA and the template DNA strand are crucial for in vitro transcription and R-loop generation. The DNA strand within hybrids can be specifically analyzed using strand-specific sequencing methods. The S9.6 antibody recognizes these hybrids with high specificity. The protocol involves generating R-loops in vitro using the pCALM3_2 plasmid, followed by enzymatic treatments and purification steps. In vitro systems often utilize synthetic hybrid spike-ins and synthetic RNA-DNA hybrids, designed using complementary DNA sequences, as controls for normalization and assay validation. Immunoprecipitation is then performed using antibody-bound magnetic beads, allowing for the capture and purification of DNA-RNA hybrids and the selective enrichment of hybrid regions for further analysis.

Thanks to Professor Frédéric Chédin’s lab at UC Davis for providing us with this protocol.

Stage 1 - R-loop preparation

Materials and reagents

25 mM rNTP stock (NEB N0466S) - dilute to 2.5 mM rNTP for experiment

T3 RNA Polymerase, 50 U/µL (NEB M0378S)

10X RNAPol Reaction Buffer

1 M DTT (from frozen stock, made in-house)

2.5% Tween-20 (diluted in water, made in-house)

pCALM3_2 plasmid (pCALM3_2 carries an R-loop forming portion of the human CALM3 gene)

RNase A, 10 mg/mL (DNase free) – dilute to 1.0 mg/mL for the experiment

RNase H, 5 U/µL (NEB M0297S)

Proteinase K, 10 mg/mL

DuRed (nucleic acid dye), 1,000X

ApaLI, 2500 units (NEB R0507S)

Nuclease free water

Steps

Mix:

pCALM3_2 - 2 µg

10X buffer - 10 µL

1M DTT - 2 µL

2.5% Tween-20 - 2 µL

2.5 mM rNTP - 10 µL

Nuclease free water to 99.4 µL total

Initiate the reaction by adding 0.6 µL of T3 RNA Polymerase, mix gently, and split into two reactions (50 µL each).

Incubate at 37°C for 10 min.

Inactivate enzyme by adding 1 µL of 10 mM EDTA.

To one sample, add 10 µL of 0.1 mg/mL RNase A, and the other (negative control) add 10 µL 0.1 mg/mL RNase A and 4 µL RNase H.

Incubate for 30 min at 37°C.

Add 4 µL of Proteinase K and incubate for 30 min at 37°C.

Clean up using an Axygen PCR purification kit and elute in 50 µL ddH₂O, separately.

For each sample (2 samples in total), split into two tubes. Put one tube on ice, and use the other for the following digestion.

Digest:

25 µl DNA
5 µL 10X Cutsmart buffer NEB
1 µL Ll ApaLI NEB
19 µL ddH₂O
50 µL total

Incubate at 37°C for 2 h.

Clean up using the Axygen PCR purification kit and elute in 25 µL ddH₂O.

You should have four samples (2–5) plus one pCALM3_2 plasmid (1) for the DRIP experiment.

To confirm that the R-loop formation has occurred, load ~200 ng of your sample on a 0.9% 1X TBE gel without nucleic acid dye and run at 90 V for 60 min.

Use 10% Glycerol as a loading dye. Post-stain with DuRed (nucleic acid dye). R-loop formation causes a characteristic shift in mobility compared to un-transcribed or RNase H-treated samples (Figure 1).

Stage 2 - DNA-RNA hybrid immunoprecipitation using antibodies pre-immobilized on beads

Materials and reagents

PBS

Triton X100 or NP-40

Salmon sperm single strand DNA (ssDNA)

Recombinant Anti-DNA:RNA hybrid antibody [S9.6] (ab234957)

Mouse IgG1, kappa monoclonal [MOPC-21] - isotype control (ab18443)

EDTA

2.5% Tween-20 (diluted in water, made in-house)

ApaLI (NEB R0507S)

RNase A, 10 mg/mL (DNase free) – dilute to 1.0 mg/mL for the experiment

RNase H, 5 U/µL (NEB M0297S)

Proteinase K, 10 mg/mL

DuRed (nucleic acid dye), 1,000X

Qiagen PCR purification kit

Steps

Prepare eight tubes of Protein A beads. Add 100 µL of protein A beads to each tube and wash twice in 1 mL of 1X PBS and 0.1% Triton X-100 (0.1% NP-40 can be used instead) by centrifuging for 1 min at the manufacturer's recommended speed at 4°C. Carefully aspirate the supernatant each time.

Resuspend the beads in each tube with 1 mL 1X PBS, 0.1% Triton X-100 (0.1% NP-40 can be used instead), and 7.5 µg ssDNA (20 µL beads), and shake gently for 10 min at room temperature.

Centrifuge for 1 min at the manufacturer's recommended speed at 4°C, then aspirate the supernatant. Wash once in 1 mL of 1X PBS, 0.1% Triton X-100 (0.1% NP-40 can be used instead), and centrifuge 1 min at the manufacturer's recommended speed at 4°C, carefully aspirate the supernatant.

Add 5 µL S9.6 antibody (1 mg/mL, test antibody 5 µg), to the beads in one tube and 5 µL isotype antibody (5 µg) to the remaining four tubes as a negative control. Make the samples up to 1 mL using 1X PBS with 0.1% Triton X-100 (0.1% NP-40 can be used instead).

Shake gently for 10 min at room temperature.

Wash twice with 1 mL 1X PBS with 0.1% Triton X-100 (0.1% NP-40 can be used instead), centrifuge for 1 min at the manufacturer's recommended speed at 4°C, and aspirate the supernatant.

Resuspend each tube in 100 µL PBS with 0.1% Triton X-100 (0.1% NP-40 can be used instead) and add 1 µL 0.5M EDTA.

Steps

Remove 5 μL of each input R-loop (RNAse A treated), R-loop (RNase A+H treated), ApaLI digested R-loop (RNase A treated), ApaLI digested R-loop (RNase A+H treated) for gel analysis.

Add 35 μL of R-loop (RNase A treated), R-loop (RNase A+H treated), ApaLI digested R-loop (RNase A treated), ApaLI digested R-loop (RNase A+H treated) to two tubes (S9.6, isotype), respectively.

Rotate gently for 2 hours at 4 °C.

Centrifuge 1 min at 3,600 rpm, 4 °C, remove all depleted supernatants, and retain for electrophoresis.

Wash three times in 1x PBS, 0.1% Triton X-100, centrifuge for 1 min at 3,600 rpm, 4 °C, and aspirate the supernatant.

Add 50 μL elution buffer + 5 μL protease K, shake for 30 mins at 1,400 rpm at 50 °C, and centrifuge for 1 min at 13,000 rpm at room temperature. Collect the supernatant.

Clean up using the Qiagen PCR purification kit and elute in 30 μL.

Figure 2. DNA-RNA hybrid Immunoprecipitation (DRIP) data. pCALM3_2 was used to generate R-loops. S9.6 (ab234957) immunoprecipitates R-loops in the presence or absence of prior digestion by ApaLI, which does not affect R-loop structure. Prior treatment with RNase A (digests single-stranded RNA) does not affect the IP signal, whereas prior treatment with RNase H (digests RNA in DNA-RNA hybrids) eliminates the signal.

Comparison to other methods

Compared to RNA immunoprecipitation (RIP) or chromatin immunoprecipitation (ChIP), DRIP offers unique advantages for studying RNA-DNA hybrids. While RIP focuses on RNA-protein interactions and ChIP targets DNA-protein complexes, DRIP specifically isolates RNA-DNA hybrids, providing insights into transcriptional regulation and genome instability. Unlike bisulfite sequencing or RNase H mapping, DRIP is less labor-intensive and offers higher specificity through antibody-based capture. It is particularly useful for detecting R-loops at specific loci or across the genome using qPCR or sequencing.

Sequencing-based methods such as DRIP-seq, RDIP-seq, and RNA-seq have further advanced the field by enabling genome-wide mapping of R-loops. These approaches can capture R loops with high resolution and strand specificity, allowing for the analysis of DNA strands and the detection of negative strand signals that indicate hybrid formation on the template strand. In these assays, ribosomal DNA regions are frequently used as internal controls for normalization due to their stable hybrid signals. By leveraging these techniques, researchers can comprehensively map and quantify R-loops across the genome, providing deeper insights into their formation, regulation, and biological significance.

Applications

DRIP is used to study transcriptional regulation, genome stability, and epigenetics. It enables the detection of R-loops, which are implicated in DNA damage, replication stress, and neurodegenerative diseases. The protocol supports both locus-specific and genome-wide analyses, making it suitable for studies involving gene expression profiling, chromatin structure, and RNA processing. DRIP is also valuable in cancer research, where aberrant R-loop formation can contribute to genomic instability and tumorigenesis.

DRIP can be applied to study R-loop formation and class switch recombination in stimulated B cells, particularly at immunoglobulin class switch regions. This approach helps elucidate how R-loops facilitate recombination events and regulate the rearrangements of immunoglobulin genes. In addition, DRIP has been used to investigate the role of R-loops in mitochondrial DNA replication and DNA replication, where these structures can interfere with the progression of the replication fork. DNA topoisomerase enzymes are often involved in resolving R-loops during these processes to maintain genome stability.

In mammalian cells, DRIP enables the analysis of nucleic acids, including the mapping of DNA: RNA hybrids and the use of genomic RNA and double-stranded DNA as spike-in controls for normalization. The technique is also useful for examining DNA methylation patterns and DNA backbone composition, providing insights into how these factors influence R-loop stability and function.

Limitations

While DRIP is a robust method, it has limitations. The specificity of the S9.6 antibody can vary depending on sample conditions, and cross-reactivity with other nucleic acid structures, such as single-stranded DNA or double-stranded DNA, may occur. Careful protocol optimization is required to ensure specificity for the correct DNA strands. The protocol requires careful optimization of enzymatic treatments and purification steps to avoid degradation or loss of hybrids. Additionally, DRIP does not provide direct information on the protein components of R-loops, limiting its use in protein-RNA interaction studies. Quantitative interpretation can also be affected by sample quality and antibody efficiency.

Troubleshooting

Common issues in DRIP experiments include low yield, poor specificity, and inconsistent results. To improve yield, ensure optimal R-loop formation by verifying plasmid transcription and enzyme activity. Use fresh reagents and RNase-free conditions to prevent degradation. Inefficient precipitation or loss of precipitated DNA during purification can also reduce the overall yield, so it is essential to carefully follow the precipitation and recovery steps. If specificity is low, confirm antibody binding with controls and optimize washing steps. Gel electrophoresis can help validate R-loop formation before immunoprecipitation. To achieve consistent results, standardize incubation times and temperatures, and utilize validated purification kits to ensure reproducibility.