In the field of epigenomics, accurate analysis of N6-methyladenosine (m6A) modification is the key to revealing the RNA regulatory network. With the technical innovation, the single-base resolution detection method has become the core tool to study the specific function of the m6A site, among which GLORI-seq, miCLIP, and Mazter-seq occupy an important position by virtue of their unique technical principles.

Based on the principle of antibody-antigen cross-linking and immunoprecipitation, miCLIP realizes single-base localization by capturing abnormal reverse transcription signals, but its performance is limited by antibody specificity and cross-linking efficiency. Mazter-seq uses the cleavage characteristics of MazF endonuclease on unmodified ACA sequences to achieve Qualcomm screening, but it cannot cover the whole genome modification sites due to sequence dependence. GLORI-seq achieves quantitative detection through a specific demethylation reaction of the ALKBH5 enzyme, combined with sequencing differences between the treatment group and the control group, and has high specificity and chemometric analysis ability.

The significant differences between the three methods in resolution, quantitative ability, applicable sequence range, and technical complexity make the matching of research objectives and method selection the key to experimental design. This paper systematically compares the technical principles, advantages, limitations, and applicable scenarios of GLORI-seq, miCLIP, and Mazter-seq, and provides a theoretical basis and practical guidance for the method selection of m6A epigenomics research.

The Need for Single-Nucleotide Resolution in Epitranscriptomics

As the most abundant modification type in eukaryotic mRNA, m6A has a profound influence on gene expression regulation, cell fate determination, and disease occurrence and development, so it is irreplaceable to locate it accurately at the single-base level.

• The necessity of single-base resolution is first reflected in the functional verification level. The position of m6A modification on mRNA directly determines its regulatory effect: m6A in the 5' untranslated region can promote the translation initiation of mRNA, while m6A in the 3' untranslated region is more related to the stability of mRNA. If the modification site cannot be located accurately, it will be difficult to establish a direct relationship between the modification and function.
• Secondly, single-base resolution is the basis of motif analysis. The occurrence of m6A modification has a certain sequence preference, and the discovery of classical RRACH(R=A/G, H=A/C/U) motif is based on the statistical analysis of a large number of precise modification sites. High-resolution site data can reveal the conservation and variation characteristics of motifs more accurately, and provide a basis for predicting new modification sites and studying the recognition mechanism of methyltransferase. Low-resolution techniques often cover up the sequence details around the site, which leads to the deviation of motif analysis.
• In addition, single-base resolution is very important for understanding allele-specific modification. In the scenario of genomic imprinting or unbalanced allele expression, m6A modification may only occur in one of the two alleles. Only through the detection of single-base level can we distinguish the modification States of different alleles and reveal the allele-specific mechanism of epigenetic regulation.

To sum up, single-base resolution is the technical basis for in-depth analysis of the biological function of m6A modification, and its development has promoted the leap from descriptive research to mechanism research of epigenomics.

Differential peak analysis allows to identify true m6A sites from miCLlP2 data (Körtel et al., 2021)

miCLIP: Principle and Trade-offs

miCLIP, as a classical technique for detecting m6A with single-base resolution, is based on the ultraviolet cross-linking between anti-m6A antibody and the modification site, and locates modification by capturing truncation or mutation signals during reverse transcription. Relying on a mature antibody system, this technology provides a feasible scheme for fine mapping of m6A, but it also faces some limitations, such as complex technical operation, dependence on antibody specificity, and cross-linking artifacts. It is very important to analyze its principles and trade-offs for the rational application of this technology.

Why miCLIP is Important

miCLIP (m6A-cross-linking-immunoprecipitation) technology is a single-base resolution m6A detection method based on antibody cross-linking and immunoprecipitation. The core principle is that a specific anti-m6A antibody binds to the m6A modification site in RNA, and covalent cross-linking between antibody and RNA is induced by ultraviolet light (usually 254nm) to form a stable RNA-antibody complex.

Subsequently, these complexes were enriched by immunoprecipitation, and RNA was reverse transcribed. In the process of reverse transcription, the cross-linking site will lead to the stagnation or error of reverse transcriptase, resulting in truncated cDNA or cDNA with specific mutation (such as C→T).

By high-throughput sequencing analysis of these cDNA, combined with bioinformatics analysis to identify truncation sites or mutation sites, the precise location of m6A modification can be determined, and single-base resolution can be achieved.

Superiority of miCLIP

• Single-base resolution: miCLIP can locate the m6A modification to a single nucleotide level by capturing the truncation or mutation signal in the reverse transcription process, which meets the requirements of fine localization.
• Relying on mature antibodies: This technology uses widely verified anti-m6A antibodies (such as ab151230, 202003, etc.). These antibodies have been proven to have high specificity in years of research, which lowers the threshold of technology development.
• Wide applicability: Theoretically, it is suitable for various RNA sample types, including mRNA, lncRNA, etc., without relying on a specific sequence background.

Limitations of miCLIP

Technical difficulty is high: the efficiency of UV-crosslinking is affected by many factors, such as crosslinking time and RNA secondary structure, and the experimental conditions need to be strictly optimized. In addition, the truncation and mutation signals produced during reverse transcription are often weak, which puts forward higher requirements for sequencing depth and data analysis methods.

• Low flux: The immunoprecipitation step takes a long time, and the antibody cost is high, so it is difficult to process a large number of samples at the same time, which limits its application in large-scale screening research.
• Still relying on antibodies: Although the specificity of antibodies is high, there may still be cross-reactions, especially cross-binding with structurally similar modifications (such as m6Am), leading to false positive results.
• Potential cross-linking artifacts: Excessive cross-linking may lead to non-specific RNA-antibody binding, or cause RNA structure changes, interfere with the reverse transcription process, and produce false positive signals.

Generally speaking, miCLIP technology provides a feasible scheme for single-base resolution detection of m6A, but it has some limitations in the complexity and flux of experimental operation, and is more suitable for fine research with a small sample size.

Methods for m6A methylation detection (Han et al., 2020)

Mazter-seq: An Endonuclease-Based Approach

Mazter-seq, as a m6A detection technology based on endonuclease, takes the sequence-specific cleavage of MazF as the core principle. The enzyme can accurately identify and cleave the unmodified ACA sequence, but does not affect the modified ACA site containing m6A, thus realizing single-base resolution detection. Its antibody independence and Qualcomm potential provide a unique tool for the study of m6A in the specific sequence background, but it is also limited by sequence dependence.

Mazter-seq for m6A detection

Mazter-seq technology is a single-base resolution m6A detection method based on endonuclease-specific cleavage, and its core tool is MazF endonuclease from Escherichia coli. MazF can specifically recognize and cleave the phosphodiester bond between A and C in an unmodified ACA sequence, but when A in the ACA sequence is modified by m6A, the cleavage activity of MazF is inhibited and the site is preserved.

The experimental process is roughly as follows: RNA is extracted and fragmented, and the MazF enzyme is added for digestion. The unmodified ACA sequence is cut into short segments, while the modified ACA sequence containing m6A remains intact. By separating and enriching uncut RNA fragments and using high-throughput sequencing, the position of m6A modification can be determined, and single-base resolution detection can be realized.

Superiority

• Single-base resolution: Because the cleavage of MazF has strict sequence specificity and only works on unmodified ACA sequences, it can accurately identify A modified by m6A in ACA sequences, and achieve single-base resolution.
• No antibody is needed: This technology is based on the specific cleavage of an enzyme, which avoids the possible cross-reaction and batch difference problems in antibody-dependent methods and improves the stability and reliability of the results.
• High multiplex ability: MazF digestion reaction conditions are relatively simple, and the cost is low, and it can process multiple samples at the same time, which is suitable for large-scale Qualcomm screening research.

Limitations

Depending on the specific sequence background, Mazter-seq can only detect the m6A modification in the ACA sequence, but it can't identify the modification sites that are not in the sequence background, which leads to the limited detection range and may miss a large number of important m6A sites.

• The output result is a binary signal: This technique can only judge whether A in the ACA sequence has been modified by m6A (that is, "yes" or "no"), and cannot provide the abundance information of modification, so it is difficult to make quantitative analysis and study the dynamic change of modification.
• Effect of enzyme digestion efficiency: The enzyme digestion efficiency of MazF is affected by reaction conditions, RNA secondary structure, and other factors. If the enzyme digestion is incomplete, the unmodified ACA sequence may be misjudged as a modification site, leading to false positive results.

To sum up, Mazter-seq technology has advantages in Qualcomm detection under a specific sequence background, but its application scope is limited due to its sequence dependence and non-quantification.

Outlines of different strategies for m6A mapping (Yin et al., 2022)

GLORI-seq: The Quantitative Enzymatic Standard

In epigenomics, the accurate quantification of m6A modification is very important to analyze its function. GLORI-seq relies on the principle of ALKBH5 enzymatic transformation to realize the quantitative detection of single-base resolution and break through the limitations of traditional methods. By comparing the difference of base conversion between enzyme-treated and untreated samples, it accurately locates the m6A site and quantifies the abundance, which has become a standard enzymatic technology with both specificity and quantitative ability, and provides a powerful tool for in-depth study on the dynamic regulation of m6A.

Principle Comparison

The core principle of GLORI-seq technology is based on an enzymatic conversion reaction, which is essentially different from the immunological principle of miCLIP and the enzymatic digestion principle of Mazter-seq.

MiCLIP relies on the specific binding of anti-m6A antibody to the modification site, and enriches the target RNA fragment through cross-linking and immunoprecipitation, and its signal comes from the interaction between the antibody and RNA and the abnormality in the subsequent reverse transcription process. Mazter-seq uses the MazF enzyme to specifically cleave the unmodified sequence and realizes detection by retaining the modified site.

While GLORI-seq uses human ALKBH5 demethylase, which can specifically catalyze the demethylation of m6A-modified adenosine and convert it into common adenosine (A). By comparing the sequencing results of RNA samples treated with ALKBH5 and untreated RNA samples, the site of base transformation can be identified, and the position of m6A modification can be determined, thus realizing single-base resolution detection.

Technical Superiority

• Chemometrics: GLORI-seq can accurately reflect the abundance of m6A modification by calculating the base conversion ratio of specific sites in the treatment group and the control group, and provide quantitative chemometrics data. This enables researchers to analyze the dynamic changes of the modification level and the modification differences between different samples, which provides more information for understanding the regulation mechanism of m6A modification.
• Comprehensive detection range: Unlike Mazter-seq, GLORI-seq does not depend on a specific sequence background, and can detect m6A-modified sites in all types of sequences, covering a wider range and avoiding site omission caused by sequence restriction.
• High specificity: ALKBH5 demethylase has high specificity for m6A, only catalyzes the demethylation reaction of m6A, and has no obvious effect on other RNA modifications (such as m5C, m6Am, etc.), which greatly reduces the risk of false positive results.

Technical Limitations

• Dependence on complete reaction efficiency: The demethylation efficiency of ALKBH5 directly affects the accuracy of the detection results. If the reaction is incomplete, the untransformed m6A site may be misjudged as an unmodified site, resulting in a false negative. Therefore, it is necessary to optimize the reaction conditions strictly to ensure the efficient enzymatic reaction.
• The requirement for RNA input is higher: Compared with Mazter-seq, GLORI-seq needs more initial RNA (usually microgram level), which may become a limiting factor in the research with limited sample size (such as clinical micro-samples).

Despite some limitations, GLORI-seq has become a potential technical method in the study of epigenomics because of its quantitative ability, comprehensiveness, and high specificity.

Schematic representation of two antibody-free methods: DART-m 6 A-Seq and Mazter-Seq (Yang et al., 2024)

Comparative Analysis: A Guide for Method Selection

Choosing the appropriate m6A detection technology should be based on the specific research objectives, combined with the characteristics of each technology, for comprehensive consideration. Based on different research requirements, the decision matrix of method selection is constructed below.

Aim for Quantitative Analysis

GLORI-seq is the best choice if the research focuses on exploring the abundance changes of m6A modification, such as the dynamic regulation of the modification level in different physiological states or disease stages. It can provide modification abundance data at the single base level, accurately quantify the chemometric characteristics of m6A by calculating transformation efficiency, and provide key information for revealing the quantitative relationship between modification and gene expression regulation.

MiCLIP and Mazter-seq have obvious deficiencies in quantitative ability: The signal intensity of miCLIP is affected by many factors, such as antibody binding efficiency and cross-linking efficiency, so it is difficult to quantify accurately; Mazter-seq can only output binary signals, and can't reflect the difference in modification abundance.

Aim for Maximum Site Coverage

GLORI-seq and miCLIP are more suitable when the research needs to fully capture the m6A modification sites within the genome, especially to explore new modification sites or study the overall distribution characteristics of modifications.

GLORI-seq is not limited by sequence background and can detect all possible m6A sites, with the widest coverage. Although miCLIP depends on antibodies, it is not limited to specific sequences, and it can detect modification sites in various RNA types. Both of them are better than Mazter-seq in site coverage, and the latter can only detect m6A modification in the ACA sequence, which will miss a large number of important sites in a non-ACA background.

Qualcomm Screening of ACA Loci

Mazter-seq has obvious advantages for the research focused on the modification of m6A in ACA sequence, such as exploring the function and regulation mechanism of modification in the background of this sequence. The operation is relatively simple, the cost is low, and the Qualcomm quantity can be detected, so it is suitable for large-scale sample screening research.

At this time, although GLORI-seq and miCLIP can also detect the modification in the ACA sequence, they are not dominant in terms of flux and cost. The enzymatic reaction and sample processing flow of GLORI-seq are relatively complicated, while miCLIP is limited by the antibody cost and the complexity of experimental operation, which are not suitable for large-scale Qualcomm screening of ACA sites.

Conclusion

m6A modified single-base resolution detection technology is a key tool to promote the in-depth development of epigenomics research. miCLIP, Master-seq, and GLORI-seq have their own characteristics, which provide diverse choices for different research needs.

How to choose right method for your research

MiCLIP technology, with its single-base resolution and the advantage of relying on mature antibodies, still has some value in the study of fine localization with a small sample size, but its technical complexity and antibody dependence limit its wide application. Mazter-seq, with its simplicity, high efficiency, and Qualcomm capacity, has performed well in the study of m6A modification in ACA sequence background, but its sequence dependence and its inability to quantify limit its application.

As a quantitative detection technology based on enzymatic transformation, GLORI-seq has obvious advantages in single-base resolution, quantitative ability, comprehensiveness, and specificity of detection, and provides a powerful tool for in-depth analysis of the biological function of m6A modification. Although there are some challenges in reaction efficiency and RNA input, with the continuous optimization of technology, these problems are expected to be solved.

In the future, with the continuous development of technology, there may be new methods combining the advantages of various technologies to further improve the accuracy, efficiency, and applicability of m6A detection. Researchers should reasonably choose technical methods according to the specific research objectives, give full play to the advantages of different technologies, promote the research of epigenomics to a higher level, and provide a new perspective and basis for understanding the regulation mechanism of life activities and the diagnosis and treatment of diseases.

References

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