The innovation of genomics technology has consistently expanded the cognitive boundaries of nucleic acid structure and function. When analyzing R-loop, a triple-stranded structure formed by DNA-RNA hybrid and single-stranded DNA, the evolution of methodology has deeply influenced the depth and breadth of research, from the early DRIP-seq to the emerging technologies such as DRPC-seq and R-ChIP in recent years. As a "double-edged sword", R-loop not only participates in physiological processes such as gene expression regulation and DNA repair, but also induces genomic instability, which is closely related to cancer and neurodegenerative diseases.

Accurately locating the distribution of R-loop and clarifying its chain specificity and dynamic changes have become the core premise to reveal its biological mechanism. This paper focuses on the DRIPc-seq technology, and through systematic comparison with DRIP-seq, S1-DRIP-seq, R-ChIP and other methods, analyzes its unique advantages and applicable boundaries in the aspects of chain specificity, resolution, operational complexity, etc., and clarifies its position in the genomics tool library, to provide reference for researchers to choose the optimal technical path according to experimental objectives and help promote the in-depth research on the biological function and disease association of R-loop.

This article systematically compares DRIPc-seq with DRIP-seq, S1-DRIP-seq, and R-ChIP in terms of strand specificity, resolution, and operational complexity, clarifies DRIPc-seq's unique position in genomic tools, and provides a guide for selecting optimal R-loop positioning technologies.

DRIP-seq: A Foundational Technique

DNA-RNA immunoprecipitations followed by sequencing (DRIP-seq) is the first technology to realize whole genome R-loop localization, which lays a key foundation for analyzing the biological function of this special nucleic acid structure. Its core principle revolves around the S9.6 monoclonal antibody, which is highly specific for DNA-RNA hybrids and can accurately identify the hybridization region in the R-loop.

This technique confirmed the non-random distribution of R-loop in the genome for the first time, and revealed that its preference was concentrated in gene promoters, transcription termination sites, and immunoglobulin loci, which provided direct evidence for the subsequent study of the relationship between R-loop and transcription regulation and DNA replication.

Although DRIP-seq is groundbreaking, its technical limitations have also become the starting point for subsequent methodological innovation. The most prominent defect is the lack of chain specificity, which makes it impossible to distinguish the template chain and non-template chain corresponding to RNA in the R-loop, and it is difficult to relate the R-loop and transcription direction.

In addition, the verification of the results is highly dependent on Southern blot, which is not only cumbersome to operate, but also covers only a few candidate regions, which limits the reliability of the whole genome data. At the same time, limited by the fragmentation strategy and enrichment method, the localization resolution of DRIP-seq is low (usually at the level of hundreds of base pairs), and it is difficult to accurately define the boundary of the R-loop.

Even so, these limitations have not weakened its value, as the first Qualcomm tool for R-loop research, DRIP-seq, has established a research paradigm in this field, and its findings provide a comparative baseline for subsequent technologies (such as DRPC-seq), which promotes the systematic exploration of the biological significance of R-loop.

The S9.6 antibody is likely to recognize also ssDNA-containing non-B DNA forms that do not correspond to R-loops (Vanoosthuyse et al., 2018)

Core Advantages of DRIPc-seq

As an upgraded technology of DRIP-seq, DRIPc-seq is a powerful tool for parsing R-loops. R-loop is closely related to gene expression and genome stability, while DRIPc-seq shows its unique core advantages in R-loop research through innovative processes.

cDNA Conversion-Driven Strand Specificity

DRIPc-seq added a key cDNA transformation process on the basis of the enrichment of R-loop by immunoprecipitation of DRIP-seq: the R-loop complex captured by S9.6 antibody was first treated with DNase I to release its RNA chain; Using this RNA as a template, the first cDNA strand was synthesized by reverse transcription with dNTP mixture containing dUTP. When the second strand is synthesized, the first strand containing dUTP is degraded by uracil-N-glycosylase (UNG), and only the second strand complementary to the original RNA remains.

This process makes the final sequenced cDNA fragment strictly correspond to the RNA template chain in the R-loop, so that the DNA chain where the R-loop is located can be determined by the direction of sequencing reading, and the chain-specific localization can be realized. This design directly solves the core problem that DRIP-seq can't distinguish the strand direction, and provides accurate data for analyzing the correlation between R-loop and transcription direction.

Technical Advantages of DRIPc-seq

In addition to chain specificity, the improvement of DRIPc-seq in genome-wide coverage and positioning accuracy has made it the mainstream technology of R-loop research, which has significantly expanded the depth and breadth of research.

• Genome-wide, unbiased discovery ability: DRIPc-seq does not need to design probes in advance, and realizes unbiased capture of all enriched R-loop RNA through random primer reverse transcription, thus avoiding the potential probe preference problem in DRIP-seq. This enables this technique to find unknown R-loop regions, especially in the research of non-coding RNA-related R-loops.
• More accurate mapping of the initial site: Through faithful replication of RNA template by cDNA synthesis, DRIPc-seq can reflect the boundary information of R-loop more accurately, and improve the positioning resolution to near the single base level. This enables researchers to analyze the exact distance between the start site of R-loop and the start site of transcription (TSS) and poly (A), and provides fine data for exploring the relationship between the formation of R-loop and the process of transcription start and termination.

R-loops and R-loop-binding proteins as biomarkers (Khan et al., 2022)

Nuclease-Enhanced Mapping via S1-DRIP-seq

S1-DRIP-seq and ssDNA-seq find another way to locate R-loop with the help of nuclease characteristics. They don't depend on antibodies, but use S1 nuclease's ability to cut single-stranded DNA to enrich related regions, which provides an important supplement for antibody-dependent technology and expands the technical choice of R-loop research.

Strategic Principle of S1-DRIP-seq

The core principle of S1-DRIP-seq is based on the specificity of S1 nuclease to cleave single-stranded DNA: there are exposed single-stranded DNA regions in the R-loop structure, while double-stranded DNA and DNA-RNA hybrids are relatively tolerant to S1 nuclease. In the experimental process:

• After denaturation and renaturation of genomic DNA, S1 nuclease was used to cut single-stranded DNA, and the fragment containing the R-loop was retained.
• Subsequently, these resistant fragments were analyzed by sequencing, and the R-loop region was indirectly located.
• ssDNA-seq is sequenced by directly capturing single-stranded DNA fragments, in which single-stranded regions related to R-loop can be specifically detected. These two techniques do not need to rely on antibodies, which provides a non-immunoprecipitation alternative for R-loop localization.

S1-DRIP-seq Versus Antibody-Based Methods

The differences in technical principles between S1-DRIP-seq and DRIP/DRIPc-seq lead to significant differences in sensitivity, specificity, and applicable scenarios, which need to be selected according to the research objectives.

• Sensitivity difference: S1-DRIP-seq may be more sensitive to some specific types of R-loops, such as R-loops with longer single-stranded DNA regions, which are more resistant to S1 nuclease and easier to be enriched. Antibody-based methods are more direct in identifying DNA-RNA hybrids and may be more sensitive to R-loops in short hybridization regions.
• Preference of enzyme activity: The cutting efficiency of S1 nuclease is influenced by the secondary structure and length of single-stranded DNA, and there is an inherent preference, which may lead to misjudgment or missed detection of some R-loop regions. In contrast, the recognition of DNA-RNA hybrids by S9.6 antibody has a certain cross reaction, but it is less affected by the structure, and the deviation is relatively controllable.
• Loss of chain information: Similar to DRIP-seq, S1-DRIP-seq, and ssDNA-seq can't provide chain-specific information and can't distinguish the transcription direction of R-loop, which is the main limitation in studying the coupling mechanism between R-loop and transcription.

BisMapR, an RNase H-based strand-specific native R-loop detection strategy (Wulfridge et al., 2021)

R-ChIP in Antibody-Free Detection

R-With the help of the RNase H1 mutant (dRH-), ChIP technology opened up an antibody-free R-loop research path. DRH retains high affinity for R-loop but has no catalytic activity, and accurate capture is achieved through a ChIP experiment, which provides a new idea for breaking through the limitation of antibody dependence.

Principle and Application of R-ChIP

R-loop chromatin immunoprecipitations (R-chip) technology uses mutant RNase H1 protein (dRH-) to achieve the specific capture of R-loop. RNase H1 is a natural DNA-RNA hybrid-binding protein. The catalytically inactivated mutant (such as D210N) retains high affinity for R-loop, but loses the activity of degrading RNA.

By expressing the labeled dRH-protein in cells, the protein can specifically bind to the R-loop in the genome, and then using the labeled antibody to carry out the ChIP experiment, enrich the bound DNA fragments, and sequence them to realize the R-loop localization. This technique has been used to verify the distribution of R-loop in various cell lines and has played an important role in revealing the relationship between R-loop and chromatin remodeling.

R-ChIP Comparisons with Other Technologies

The specificity and resolution of R-ChIP make it complementary to antibody and nuclease technology, but its technical complexity also limits its wide application.

• Specificity: dRH-protein is highly specific for the recognition of DNA-RNA hybrids. Compared with the S9.6 antibody, its cross-reaction with double-stranded DNA and single-stranded RNA is lower, which significantly reduces the false positive signal, especially in the detection of low-abundance R-loops.
• Resolution: R-ChIP adopts the Chip process, and can achieve high localization resolution by accurately capturing the tag protein with antibodies, which is usually better than DRIP-seq and close to the level of DRPC-SEQ, and is suitable for studying the fine correlation between R-loop and chromatin protein binding sites.
• Complexity: R-ChIP needs to construct a cell line that stably expresses labeled dRH-protein, which is time-consuming and may interfere with the physiological state of cells; In contrast, DRIP-seq and DRPC-SEQ can directly use wild-type cells, which is simpler and more applicable.

BisMapR reveals strand-specific R-loop formation across a subset of enhancersin mESCs (Wulfridge et al., 2021)

Decision Guide for Experimental Setup

In the research on R-loop, it is very important to choose the appropriate positioning technology. Different R-loop positioning technologies differ in many core characteristics, which directly affect the reliability and applicability of experimental results.

Resolution Ratio

Resolution refers to the ability of technology to accurately determine the position of the R-loop on the genome, usually in base pairs. Higher resolution can define the starting and ending sites of R-loop more accurately, which is helpful to study its interaction with other short sequence regulatory elements.

• The resolution of DRIP-seq is relatively low, usually above several hundred base pairs. This is because there is no precise capture step of the R-loop boundary in its technical process, so it is difficult to accurately distinguish the specific range of the R-loop, and there are obvious shortcomings in studying the correlation between the R-loop and some short sequence control elements.
• DRIPc-seq faithfully replicates RNA template through cDNA synthesis, which can reflect the boundary information of R-loop more accurately and improve the positioning resolution to near the single base level. This allows researchers to analyze the exact distance between the start site of R-loop and the transcription start site, poly (A) site, etc., and provides detailed data for further discussion on the formation mechanism of R-loop.
• The resolution of S1-DRIP-seq is at a medium level. Because it relies on S1 nuclease to cleave single-stranded DNA to enrich the R-loop region, and the cleavage efficiency of the enzyme is affected by many factors, it is not as accurate as DRIPc-seq, but better than DRIP-seq.
• R-ChIP adopts the ChIP process, and the tag protein can be accurately captured by an antibody, which can achieve high positioning resolution, usually close to the level of DRIPc-seq. The high resolution makes it suitable for studying the fine correlation between R-loop and chromatin protein binding sites.

Specificity

Specificity refers to the ability of technology to avoid nonspecific binding when capturing R-loop, that is, whether the real R-loop can be accurately distinguished from other nucleic acid structures, which directly affects the reliability of experimental results. ​

• The specificity of DRIP-seq is at a medium level. Although the S9.6 antibody has a certain affinity for DNA-RNA hybrids, it may still have nonspecific binding with double-stranded DNA and single-stranded RNA in practical experiments, resulting in false positive signals.
• DRIPc-seq is improved on the basis of DRIP-seq, and its specificity is relatively high. The cDNA conversion step further screened the nucleic acid fragments related to R-loop, which reduced the interference caused by non-specific binding and improved the accuracy of the results.
• The specificity of S1-DRIP-seq is also moderate. The cutting efficiency of S1 nuclease is influenced by the secondary structure and length of single-stranded DNA, and there is an inherent preference, which may lead to some non-R-loop single-stranded DNA regions being misjudged as R-loop regions, thus affecting the specificity.
• R-ChIP is highly specific. The catalytic inactivation mutant RNase H1 protein (dRH-) is highly specific to DNA-RNA hybrids, and its cross-reaction with double-stranded DNA and single-stranded RNA is extremely low, which significantly reduces the false positive signal, especially in the detection of low-abundance R-loops.

Results for sample SRR5427884 (Gaspar et al., 2018)

Operational Complexity

• The complexity of operation involves the complexity of the technical process, the required experimental skills, and whether special cell lines or experimental materials are needed, which will affect the popularization and application scope of the technology.
• The operation complexity of DRIP-seq is low. Its process is relatively simple; it does not need complex enzyme treatment steps and special cell lines, and ordinary laboratories can carry out experiments after simple training, so it has been widely used in early R-loop research.
• The operational complexity of DRIPc-seq is moderate. Compared with DRIP-seq, it adds some key steps, such as cDNA transformation, which requires more detailed experimental operation and strict control of reaction conditions, but it still does not need to construct a special cell line, which can be mastered by most laboratories.
• The operational complexity of S1-DRIP-seq is also at a medium level. The core step is to cut single-stranded DNA by S1 nuclease, which requires precise control of enzyme concentration, reaction time, and temperature. The operation difficulty is slightly higher than that of DRIP-seq, but lower than that of R-ChIP.
• The operation complexity of R-ChIP is high. It needs to construct a cell line that stably expresses labeled dRH-protein, which is time-consuming and requires high experimental skills, and may interfere with the physiological state of cells, limiting its application in some laboratories.

Comparison between DRIPc-seq and related technologies

Conclusion

DRIPc-seq occupies a unique position in the genomics tool library: it inherits the whole genome analysis ability of DRIP-seq, and makes up for the key defects through the breakthrough of chain specificity, while avoiding the dependence of R-ChIP on stable cell lines and the enzyme preference of S1-DRIP-seq. Its balanced resolution, specificity, and operational feasibility make it the first choice for most R-loop global mapping studies.

Although it needs to give way to R-ChIP in the extremely specific demand scenario, it does not need to preset the unbiased and chain information retention ability of the probe, which still makes it irreplaceable in analyzing the relationship between R-loop and transcription dynamics and genome stability, and provides accurate and flexible technical support for exploring the dual biological functions of R-loop.

References

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3. Wulfridge P, Sarma K. "A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide." Elife. 2021 10: e65146.
4. Gaspar JM. "Improved peak-calling with MACS2." bioRxiv. 2018 12: 17.