R-loop, as a triple-stranded nucleic acid structure composed of DNA-RNA hybrid and single-stranded DNA, plays a key role in the dynamic change of the genome. Under physiological conditions, they participate in key processes such as gene expression regulation, DNA replication initiation, and damage repair, and influence the fate of cells by shaping chromatin structure. However, when the R-loop is out of balance, its abnormal accumulation will become the inducement of genome instability, leading to replication fork stagnation, DNA breakage, and even chromosome translocation, which is closely related to cancer, neurodegenerative diseases, and other diseases.

Analyzing the genome distribution of R-loop is the premise to understand its dual functions. Although the traditional DRIP-seq technology started the Qualcomm era of R-loop research, it was limited by the lack of chain specificity and insufficient resolution. The emergence of DRIPc-seq has achieved a technical breakthrough. By introducing the cDNA transformation step, not only is the chain information retained, but also the detection sensitivity and positioning accuracy are significantly improved, which provides a brand-new tool for drawing high-resolution R-loop maps and pushes us into a new stage of R-loop functional mechanism research.

This article details R-loops' dual nature in genome biology, explains how DRIPc-seq overcomes DRIP-seq's limitations via cDNA transformation, outlines its step-by-step protocol, and highlights its value for R-loop research and disease studies.

The Dual Nature of R-loops in Genome Biology

R-Loop, as a triple-stranded structure formed by DNA-RNA hybrid and single-stranded DNA, plays a dual role in the genome. It can not only regulate gene expression, participate in DNA replication and repair, but also maintain genome homeostasis. Its abnormal accumulation will lead to replication fork stagnation and DNA breakage, threatening the stability of the genome, which is closely related to many diseases. It is of great significance to analyze its distribution mechanism for biological research.

Basic Biological Characteristics of R-loop

R-loop is a triple-stranded nucleic acid structure consisting of a DNA-RNA hybrid and a single-stranded DNA, which widely exists in genomes from prokaryotes to eukaryotes. In mammalian cells, about 5% of genomic regions will form stable R-loop structures, which are not randomly distributed but tend to be enriched in genomic regions with important functions such as gene promoters, terminators, enhancers, and immunoglobulin loci.

The formation of R-loops is usually closely related to the transcription process. When RNA molecules produced by RNA polymerase transcription fail to separate from template DNA in time, but recombine with template DNA to form a hybrid, the non-template DNA will be exposed as a single strand, and then the R-loop structure will be formed.

Physiological Function of R-loop

Under normal physiological conditions, the R-loop plays an important regulatory role and participates in key biological processes such as gene expression regulation, DNA replication initiation, and DNA damage repair.

• In terms of gene expression regulation, R-loop affects transcription activity through various mechanisms. R-loop formed in the gene promoter region can regulate the transcription initiation of genes by recruiting transcription-activating factors or inhibiting factors.
• In the process of DNA replication, R-loops can not only be used as a regulatory signal for the initiation of replication, but also affect the process of replication fork. Some studies show that the R-loop in a specific region can recruit replication initiation complexes and promote the initiation of DNA replication. At the same time, the R-loop may interact with the replication fork to adjust the moving speed of the replication fork and ensure the accuracy of DNA replication.
• R-loop plays a dual role in DNA damage repair. On the one hand, R-loop can be used as a recognition signal of DNA damage, recruiting repair-related proteins to the damage site and starting the repair process; On the other hand, the R-loop structure itself may also participate in the regulation of the repair mechanism.

Functions of R-loops (Li et al., 2023)

From DRIP-seq to DRIPc-seq: Overcoming Limitations

In order to analyze the genome distribution of R-loop, DRIP-seq enriched DNA-RNA hybrids with S9.6 antibody, which became an important tool in the early stage. However, it has some limitations, such as probe dependence, lack of chain specificity, and antibody deviation, which restrict the depth of research. DRIPc-seq came into being. By introducing some improvements, such as cDNA transformation, these bottlenecks were broken, and R-loop research entered a new stage.

Immunoprecipitation Principle Based on the S9.6 Antibody.

S9.6 antibody is a monoclonal antibody that can specifically recognize DNA-RNA hybrids. It has high affinity for DNA-RNA hybrids, but very low affinity for double-stranded DNA and single-stranded RNA, which makes it an ideal tool for separating and enriching R-loop structures. Immunoprecipitation technology based on S9.6 antibody is the core principle of DRIP-seq and DRPC-seq, and its basic process is as follows:

• Firstly, the genomic DNA is properly treated to keep the structure of R-loop intact
• Then, the antibody S9.6 is added, which specifically binds to DNA-RNA hybrid in R-loop
• The R-loop complex bound to the antibody is separated by an immunoprecipitation reaction.
• Finally, the enriched R-loop-related DNA was sequenced and analyzed to determine the position of the R-loop in the genome

Limitations of DRIP-seq

Although DRIP-seq provides an important means for R-loop genome mapping, this technology has the following obvious limitations:

• Probe dependence: DRIP-seq needs to use probes for hybridization capture in the process of library preparation, which may lead to the deviation of capture efficiency for some R-loop regions and affect the accuracy and completeness of the results.
• Lack of strand specificity: DRIP-seq can't distinguish the DNA strand from which RNA in the R-loop comes, so it can't determine the relationship between R-loop and gene transcription direction, which limits the in-depth analysis of R-loop function.
• Antibody bias: Although the S9.6 antibody has high specificity for DNA-RNA hybrids, there may still be nonspecific binding in actual experiments, resulting in false positive signals. At the same time, the affinity difference of antibodies may also lead to different enrichment efficiency for different R-loop structures, which will affect the reliability of the results.
• Limited resolution: the sequencing resolution of DRIP-seq is relatively low, so it is difficult to accurately determine the boundary and fine structure of R-loop, which is not conducive to studying the interaction between R-loop and other genomic elements.

cDNA Transformation to Achieve Higher Resolution

In order to overcome the limitations of DRIP-seq, researchers developed DRIPc-seq (DRIP with cDNA sequencing) technology. On the basis of DRIP-seq, this technology has made a key improvement and introduced the cDNA transformation step, which realized the transformation from DNA sequencing to cDNA sequencing, thus significantly improving the resolution and specificity of the technology.

In DRIPc-seq, after the R-loop complex was obtained by immunoprecipitation with S9.6 antibody, the DNA in it was not directly sequenced, but the hybrid was first treated with DNase I to release RNA chains. Then, using the released RNA as a template, a reverse transcription reaction was carried out with reverse transcriptase and dUTP to synthesize the first strand of cDNA.

Then, the second strand was synthesized and treated with uracil-N-glycosylase (UNG) to remove the strand containing dUTP, ensuring that the final library was only from the original RNA template strand. Through this process, DRIPc-seq not only retains the information of the chain and realizes the chain specificity analysis, but also improves the detection sensitivity of low-abundance R-loop through the synthesis and enrichment of cDNA, thus achieving higher resolution.

Profiling R-loops (RNA-DNA hybrids) (Guh et al., 2020)

DRIPc-seq Protocol: A Step-by-Step Workflow

As a key genome regulatory structure, the abnormality of the R-loop is closely related to many diseases. With its high specificity and chain resolution, DRIPc-seq technology has become a powerful tool for analyzing R-loop distribution. The step-by-step experimental process of DRIPc-seq is described in detail below, which provides standardized operation guidelines for accurately locating R-loop and exploring its biological functions.

Cell Lysis and DNA Extraction

Cell lysis and genomic DNA extraction are the first steps of the DRIPc-seq experiment, and the key is to operate under non-denaturing conditions to maintain the structural integrity of the R-loop. The specific steps are as follows:

• Cell collection: Collect the cells in a logarithmic growth period, wash the cells with precooled PBS 2-3 times, and remove the culture medium components.
• Cell lysis: Use a lysis buffer containing mild detergent (such as NP-40) to lyse cells, and the buffer does not contain denaturant (such as SDS, urea, etc.) to avoid damaging the R-loop structure. The lysis process is carried out on ice, which usually needs to be incubated for 30 minutes to 1 hour to ensure that the cells are completely lysed.
• Protein removal: Protease K was added to the lysate and incubated at 37℃ for several hours to degrade protein and release genomic DNA.
• Genomic DNA extraction: Using phenol-chloroform extraction or a commercial DNA extraction kit to extract genomic DNA. Attention should be paid to avoid violent oscillation in the extraction process to prevent DNA breakage. At the same time, DNA was purified by ethanol precipitation and other steps to remove residual protein and other impurities.
• Detection of DNA quality: Use agarose gel electrophoresis to detect the integrity of DNA, to ensure that the length of DNA fragments is large and there is no obvious degradation. Determine the concentration and purity of DNA by ultraviolet spectrophotometer (A260/A280 ratio should be between 1.8 and 2.0).

Enzymatic Fragmentation and S9.6 Immunoprecipitation

• Enzymatic fragmentation: The extracted genomic DNA is digested with restriction enzymes to fragment the DNA. Select appropriate restriction endonucleases (such as Hind III, EcoR I, etc.). The recognition sequences of these enzymes are widely distributed in the genome and can cut DNA into fragments with a length of about 100-500 bp, which is convenient for subsequent immunoprecipitation and sequencing analysis. The enzyme digestion reaction is carried out under suitable buffer and temperature conditions. By controlling the amount of enzyme and reaction time, the enzyme digestion is complete, and the fragment size meets the requirements. After the digestion, the restriction endonuclease was inactivated by heating.
• Pre-cleaning: In order to reduce non-specific binding, the digested DNA fragments were incubated with Protein A/G agarose beads at 4℃ for 1-2 hours for pre-cleaning. Then the supernatant was collected by centrifugation to remove DNA and impurities that were not specifically bound to beads.
• S9.6 antibody incubation: Add S9.6 antibody to the pre-cleared supernatant, and incubate it overnight with gentle shaking at 4℃ to make the antibody fully combine with the DNA-RNA hybrid in the R-loop.
• Immunoprecipitation: Add Protein A/G agarose beads, and continue to incubate at 4℃ for 2-4 hours to bind the antibody-antigen complex to the beads. Subsequently, beads were collected by centrifugation and then washed several times with washing buffer to remove unbound DNA and other impurities. The washing process should be handled gently to avoid the loss of beads.

Effect of transcription and repeat length on FMR1 R-loop formation (Loomis et al., 2014)

RNase H Treatment

In order to verify the specificity of DNA-RNA hybrids obtained by immunoprecipitation, RNase H treatment on magnetic beads is needed as a control experiment. RNase H is an enzyme that can specifically degrade RNA chains in DNA-RNA hybrids, but has no effect on double-stranded DNA and single-stranded RNA. The specific steps are as follows:

• Beads obtained by immunoprecipitation were divided into two groups. One group was used as the experimental group without RNase H treatment. The other group was treated with RNase H as the control group.
• RNase H and corresponding reaction buffer were added to beads in the control group, and incubated at 37℃ for 1-2 hours, so that RNase H could degrade the RNA chain in the DNA-RNA hybrid.
• After the treatment, the subsequent DNA extraction steps were carried out for the two groups of beads, and the DNA content and sequencing results in the two groups of samples were compared. If the enriched DNA in the experimental group mainly comes from R-loop structure, the content of DNA in the control group should be significantly reduced after RNase H treatment, and the R-loop-related signals in the sequencing results will also be significantly weakened, thus verifying the specificity of the experiment.

Ensuring Specificity and Reproducibility for DRIPc-seq

Comparing DRIPc-seq with other related technologies (such as ChIP-seq and RNA-seq) will help to evaluate the performance and advantages of DRIPc-seq more comprehensively. The specific comparison is as follows:

Comparison with ChIP-seq

• Principal difference: ChIP-seq is mainly used to study the interaction between protein and DNA, and the DNA fragments bound to the target protein are enriched by specific antibodies and sequenced; DRIPc-seq focuses on the localization of R-loop structure, based on the specific recognition of DNA-RNA hybrids by S9.6 antibody.
• Application: ChIP-seq is widely used in the study of transcription factor binding sites and histone modification. DRIPc-seq is mainly used to study the genome distribution and function of the R-loop.
• Complementarity: the two have a certain complementarity. For example, the relationship between R-loop and chromatin state can be discussed by combining DRIPc-seq data with histone-modified ChIP-seq data. Integrating with the ChIP-seq data of transcription factors, we can study the effect of R-loop on transcription factor binding.

Examples of MapReduce-based (a) and Spark-based (b) big data bioinformatics analysis frameworks (Yang et al., 2017)

Comparison with RNA-seq

• Principal difference: RNA-seq is used to study gene expression level and transcript structure by sequencing all RNA in cells; DRIPc-seq is used to sequence the DNA region corresponding to RNA in the R-loop, reflecting the distribution of the R-loop.
• Relevance: The formation of R-loop is closely related to the transcription process, so there is a certain correlation between DRIPc-seq data and RNA-seq data. By comparing the two datasets, we can analyze the relationship between the distribution of R-loop and gene expression level, such as whether the promoter or terminator region of a highly expressed gene is more likely to form R-loop.
• Uniqueness: DRIPc-seq can provide structural information of R-loop that RNA-seq can't get, while RNA-seq can explain the results of DRIPc-seq at the transcription level. The combination of the two can reveal the mechanism of R-loop in gene expression regulation more deeply.

Advantages of DRIPc-seq

• Higher specificity and resolution: Compared with DRIP-seq, DRPC-SEQ improves the specific recognition and positioning resolution of R-loop by introducing a cDNA transformation step, and can determine the boundary and chain information of R-loop more accurately.
• Synergy with other technologies: DRIPc-seq can be used in collaboration with ChIP-seq, RNA-seq, and other technologies to reveal the regulation mechanism of the genome from different levels and provide a more comprehensive perspective for the study of complex biological processes.

DRIPc-seq identifies RNase H-resistant but exosome-sensitive transcripts in fission yeast (Hartono et al., 2018)

Conclusion

From the comparison of the above benchmarks, it can be seen that DRIPc-seq, as a technology specially used for R-loop localization, has its unique advantages and application value, and complements other technologies to jointly promote the in-depth development of genome biology research.

To sum up, the emergence of DRIPc-seq technology provides a powerful tool for the research of R-loop, which overcomes the limitations of DRIP-seq and realizes the accurate and high-resolution positioning of R-loop. With the continuous improvement of technology and wide application, DRIPc-seq will play an increasingly important role in revealing the biological function of R-loop and clarifying its relationship with diseases, providing new ideas and targets for the diagnosis and treatment of related diseases.

References

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