N4-acetyl cytidine (ac4C), as a key epigenetic modification of mRNA, participates in important biological processes such as cell stress response and tumorigenesis by regulating mRNA stability and translation efficiency, and its genome-wide map analysis becomes the core premise to reveal the modification function. However, the low abundance (about 0.1% of total cytidine), chemical instability, and structural similarity with other acetylated modifications make the detection of ac4C face severe challenges.

Under this background, two mainstream detection technologies have gradually developed and matured: acRIP-seq is based on the immunoprecipitation principle of anti-ac4C antibody, and Qualcomm quantity detection is realized by enriching modified RNA fragments, which has become the main tool for early research because of its simple operation and strong compatibility. ac4C-seq relies on chemical labeling strategies (such as base conversion mediated by NaCNBH3 reduction) to transform the modified site into a mutation signal that can be recognized by sequencing, so as to achieve accurate positioning of single nucleotide resolution.

The principal difference between the two technologies leads to unique advantages and limitations: acRIP-seq is limited by antibody specificity (possibly cross-identifying other acetylation modifications), but it is suitable for low initial samples. ac4C-seq can avoid the deviation of antibody dependence, although the reaction conditions need to be strictly optimized to avoid RNA degradation. With the deepening of research, how to choose the technology according to the experimental objectives and how to integrate the data of the two to analyze the function of ac4C has become a key issue in the field.

This article compares ac4C-seq and acRIP-seq in N4-acetylcytidine RNA profiling, covering their principles, workflows, strengths, limitations, applications, technical challenges, solutions, and future directions.

Introduction to ac4C RNA Modification and Detection

As a key RNA modification, N4-acetyl cytidine (ac4C) regulates mRNA stability and translation, and is associated with cancer, viral infection, and other diseases. However, its low abundance, chemical instability, and antibody cross-reactivity bring challenges to accurate localization, which promotes the evolution of detection methods from acRIP-seq to ac4C-seq.

Biological Significance of ac4C

As a key RNA modification, ac4C is of great significance in life activities. It affects the stability of mRNA, prolongs the existence time of mRNA in cells, and provides a stable template for protein synthesis. At the same time, ac4C can improve the translation efficiency of mRNA and ensure that the cells synthesize protein efficiently. In the field of diseases, ac4C is closely related to cancer and viral infection. In esophageal squamous cell carcinoma, ac4C modification can promote the invasion and metastasis of cancer cells. In the process of virus infection, it may participate in the regulation of virus gene expression and the maintenance of the virus life cycle.

Challenge of ac4C Positioning

There are many challenges in the positioning of ac4C:

• Firstly, the abundance of ac4C in RNA is low, which makes it difficult to detect its signal and requires a highly sensitive detection method.
• Secondly, ac4C is chemically unstable, and it is easy to degrade or change its structure during the experimental treatment, which affects the accuracy of the test results.
• Thirdly, the cross-reactivity of antibodies is also a big problem. Antibodies used to detect ac4C may bind nonspecifically with other RNA modifications, leading to false-positive results.

With the development of research, the detection methods of ac4C have been developed continuously. In the early days, antibody-based methods, such as acRIP-seq, were mainly used to enrich ac4C modified RNA fragments with specific antibodies for analysis. With the development of technology, the modern chemical sequencing method, ac4C-seq, came into being. Through chemical transformation, ac4C produced detectable features, and the location and analysis of ac4C were realized.

ac4C mapping through antibody-based enrichment and sequencing (Schiffers et al., 2024)

Methodological Comparison: acRIP-seq vs. ac4C-seq

acRIP-seq and ac4C-seq are two core technologies for analyzing ac4C modification. The former relies on antibody enrichment, and its operation is simple but limited by antibody specificity. The latter realizes high-resolution detection through chemical transformation, but it requires strict experimental conditions. Both of them have their advantages and disadvantages. It is very important to compare their principles and characteristics systematically for the selection of research methods and the interpretation of results.

acRIP-seq

• A.Principle
  • a) The principle of acRIP-seq is based on the enrichment of antibodies, and anti-ac4C antibodies are used to specifically bind ac4C-modified RNA fragments, thus separating them from complex RNA mixtures.
• B. Procedure
  • a) Firstly, the whole RNA is fragmented by physical or chemical methods, and the long-chain RNA is degraded into short fragments of 100-300nt, which can not only ensure the accuracy of sequencing, but also be suitable for subsequent immunoprecipitation operations. In the process of fragmentation, specific RNase inhibitors are usually added to prevent non-specific degradation.
  • b) Secondly, mix the fragmented RNA with highly specific anti-ac4C monoclonal antibody, which is closely bound to the RNA fragment containing ac4C modification through an antigen-antibody specific recognition mechanism. The mixed system was incubated with gentle shaking at 4℃ to promote full combination.
  • c) Then, Protein A/G magnetic beads were used to capture the antigen-antibody complex, and after several rounds of rigorous buffer washing, the target RNA fragment was efficiently enriched by magnetic frame centrifugation.
  • d) Finally, the library of the enriched RNA fragments was constructed: first, the specific linker sequence was added at the end of the RNA, and then the cDNA was generated by reverse transcription, and the library was amplified and homogenized by PCR amplification technology.
  • e) A high-throughput sequencing platform was used for deep sequencing of the constructed library, and finally, bioinformatics tools were used for comparative analysis, peak identification, and functional annotation, so as to achieve accurate location and quantitative analysis of ac4C modification sites at the whole transcriptome level.
• C. Advantages and disadvantages
  • a) The advantage of acRIP-seq lies in its good compatibility with the standard RIP experimental scheme, and researchers can make use of the existing RIP experimental platform and experience to carry out research. At the same time, it requires less RNA input and is suitable for the case of a limited sample size.
  • b) However, acRIP-seq also has limitations, the most important of which is antibody specificity. The anti-ac4C antibody may cross-react with other RNA modifications, resulting in nonspecific binding, thus affecting the accuracy of the detection results and causing false-positive peaks.

Two methods used to detect ac4C modifications in RNA (Zhang et al., 2014)

ac4C-seq

• A. Principle
  • a) The principle of ac4C-seq is to transform ac4C modification into detectable features through chemical transformation. Using chemical reactions such as NaCNBH3 reduction, ac4C has specific changes, which can be identified in the subsequent sequencing process.
• B. Procedure
  • a) Firstly, RNA samples are treated with highly specific chemical probes, which can accurately target ac4C sites, and make ac4C undergo specific chemical transformation through a unique chemical reaction mechanism, so as to transform its structural modification characteristics into detectable molecular signals.
  • b) Then enter the reverse transcription reaction. Based on the recognition difference of chemically modified bases by reverse transcriptase, the chemically transformed ac4C will interfere with the normal base pairing process, resulting in characteristic mutations in the reverse transcription process and forming a cDNA library containing the position information of ac4C.
  • c) Finally, with the help of a professional bioinformatics analysis platform, high-throughput sequencing data are deeply mined by using advanced analysis tools such as the Hidden Markov Model (HMM) and sliding window algorithm.
  • d) The ac4C enrichment region is accurately located by a peak identification algorithm, and the high-precision identification and functional annotation analysis of ac4C loci are realized by combining a statistical test and a machine learning model, so as to systematically analyze the distribution map and regulation law of ac4C at the transcription level.
• C. Advantages and disadvantages
  • a) The advantage of ac4C-seq lies in its higher resolution and specificity. Based on the principle of chemical transformation, the deviation caused by the antibody is reduced, and the ac4C modification site can be located more accurately.
  • b) However, ac4C-seq also has some shortcomings. It needs optimized reaction conditions, such as pH value and temperature, which will affect the efficiency and specificity of chemical transformation. At the same time, the chemical treatment process may lead to RNA degradation and affect the experimental results.

Comparison between acRIP-seq and ac4c-seq

Applications and Biological Insights

ac4C-seq and acRIP-seq have their emphases in biological research. AcRIP-seq provides an efficient screening method for the early exploration of ac4C distribution, and ac4C-seq is analyzed by means of a high-resolution assistance mechanism. The application of ac4C and AC4C promotes the understanding of the role of AC4C in cell pathways, virus life cycle, and stress response, and lays a foundation for revealing its biological functions.

Application of acRIP-seq in the Discovery Stage

• acRIP-seq played an important role in the early research of ac4C. It is used to locate ac4C in yeast and human cells, which helps researchers to understand the distribution of ac4C in different species of cells.
• acRIP-seq revealed that ac4C was enriched in ribosome biogenesis and other pathways, which laid a foundation for further study on the functions of ac4C in these pathways.

Application of ac4C-seq in Mechanism Research

• ac4C-seq is outstanding in mechanism research because of its high resolution and specificity. In the research of viral RNA, such as SARS-CoV-2, ac4C-seq has achieved ac4C localization with single-nucleotide resolution, which is helpful to understand the mechanism of ac4C modification in viral RNA.
• In the dynamic ac4C spectrum analysis under stress conditions, such as heat shock conditions, ac4C-seq can detect the dynamic changes of ac4C modification, which provides a powerful tool for studying the function of ac4C in cell stress response.

Integration Method

The combination of acRIP-seq and ac4C-seq is an effective research strategy. acRIP-seq can be used for large-scale screening, and it can quickly find the RNA regions that may be modified by ac4C. Then ac4C-seq is used to verify and accurately locate these areas, so as to improve the accuracy and efficiency of the research. This integration method can give full play to the advantages of the two technologies and promote the in-depth development of ac4C research.

Peak counts depend on sequencing depth (Landt et al., 2012)

Technical Challenges and Solutions

Although ac4C-seq technology has promoted the modification research, the antibody specificity of ac4C-seq, batch difference, and chemical efficiency fluctuation of ac4c-seq still restrict the data reliability. To solve these technical challenges, it is necessary to optimize the experiment and innovate the calculation method to lay the foundation for accurately analyzing the function of ac4C.

• A. Defects and Solutions of acRIP-seq
  • a) acRIP-seq has some defects, among which the antibody batch variability is one. Different batches of antibodies may have differences in specificity and affinity, which affect the repeatability of experimental results. In order to solve this problem, researchers can strictly test the quality of different batches of antibodies, select antibodies with stable performance for experiments, and increase the number of repetitions in the experimental design to reduce the impact of batch differences.
  • b) In addition, there are noises caused by nonspecific peaks in the bioinformatics analysis of acRIP-seq. Researchers can reduce the interference of nonspecific peaks by optimizing the bioinformatics analysis process, such as adopting stricter peak identification standards and filtering the control experimental data. At the same time, optimizing immunoprecipitation conditions, such as adjusting antibody concentration and incubation time, can also reduce nonspecific binding and noise.
• B. Optimization of ac4C-seq
  • a) The chemical reaction efficiency of ac4C-seq is affected by many factors, such as pH value and temperature. Researchers need to determine the best reaction conditions through a large number of preliminary experiments to improve the efficiency and specificity of chemical transformation. For example, the pH value of the reaction system is accurately controlled in a suitable range, and the appropriate reaction temperature and time are selected to ensure that ac4C can be efficiently and specifically transformed.
  • b) In terms of computing tools, there are some special tools, such as AC-tools, for analyzing the mutation characteristics of ac4C-seq data. These tools can identify and analyze the mutation signals in ac4C-seq data more accurately and improve the accuracy of data interpretation. Researchers should choose and use these tools reasonably and constantly develop and optimize new calculation methods.
• C. Emerging Alternative Methods
  • a) Nanopore direct RNA sequencing technology is a new alternative method for ac4C detection, which can directly sequence RNA and detect its modification without antibody or chemical treatment. This method has the advantages of real-time sequencing and long reading length, which provides a new idea for the detection of ac4C. However, at present, the sensitivity and accuracy of ac4C detection need to be further improved, and more research and optimization are needed.

Categories of tools in the scRNA-tools database (Zappia et al., 2018)

Conclusion

ac4C-seq and acRIP-seq, as the core technologies for analyzing N4-acetyl cytidine modification, have their irreplaceable values: acRIP-seq is suitable for preliminary screening and large-scale mapping with low input requirements and mature processes, while ac4C-seq is a key tool for mechanism verification with single base resolution and high specificity.

In the future, it is necessary to unify the comparability of the two results through standardized benchmark data sets, and at the same time, promote technical integration, such as quickly locking candidate regions with acRIP-seq, accurately analyzing modification sites with ac4C-seq, and assisting with emerging means such as nanopore sequencing to break through the existing limitations. This multi-technology collaboration will deepen the understanding of dynamic regulation of ac4C in physiology and pathology, and open up a new path for targeted RNA-modified disease treatment.

References

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