In the study of epigenomics, the precise location of N-methyladenosine (m6A) modification is the core link to analyze the regulation of RNA function. Traditional detection technologies, such as MeRIP-seq, have some limitations, such as low resolution and insufficient specificity etc., but Global Marna Methylation Profiling by Enzymatic Conversion and Sequencing (GLORI-seq) has become the key technology to break through this bottleneck by virtue of the innovative principle of enzymatic transformation. Its core mechanism lies in utilizing the specificity of human ALKBH5 demethylase, that is, the enzyme can efficiently catalyze the demethylation of m6A to adenosine (A), and by comparing the sequencing differences between the enzyme-treated group and the untreated group, the single-base resolution of m6A loci can be identified. This principle not only avoids the interference of antibody cross-reaction but also improves the reliability of the results by directly detecting the base conversion signal.

The experimental process of GLORI-seq can be condensed into three key steps, which together form the technical basis of high specificity and high resolution:

• Firstly, RNA fragmentation and grouping treatment, and uniform fragments are obtained by ultrasonic or enzyme digestion and divided into enzyme treatment group and control group.
• Secondly, the demethylation reaction mediated by ALKBH5 in vitro completed the selective transformation of m6A in a specific buffer system.
• Finally, the construction of a sequencing library and data analysis, through high-throughput sequencing and comparative analysis, allows for the accurate identification and quantification of m6A loci to be realized.

These three steps are closely linked, which not only embody the core innovation of enzymatic transformation but also ensure the reliability of the detection results, making it show significant advantages in functional genomics research.

The article elaborates on GLORI-seq's core principles based on ALKBH5-mediated demethylation and its three detailed steps (RNA fragmentation, in vitro demethylation, library construction and analysis), along with its comparative advantages in m6A detection.

The Need for Precise Epitranscriptome Mapping

As the core research content of epigenetics, RNA modification plays a vital role in biological processes such as gene expression regulation, cell differentiation, and disease occurrence. Among them, m6A is one of the most abundant internal modifications in eukaryotic mRNA, and its biological significance has been widely confirmed.

m6A modification is involved in many physiological and pathological processes, such as embryonic development, stem cell differentiation, tumorigenesis, and development by affecting the stability of mRNA, translation efficiency, splicing processing, and nuclear output.

• In embryonic stem cells, m6A modification can maintain the pluripotency of stem cells by regulating the expression of pluripotency-related genes.
• In tumor cells, the abnormal expression of m6A modification-related enzymes will lead to the expression disorder of downstream target genes, and then promote the proliferation, invasion, and metastasis of tumors.

Therefore, it is of great significance to draw the map of m6A modification accurately and make clear its distribution and dynamic changes in the genome, so as to deeply understand its biological functions.

However, there are many limitations in the early mapping technology of m6A modification, which restrict the in-depth development of related research. Among them, MeRIP-seq/m6A-seq is the earliest widely used technology. Its principle is to immunoprecipitate RNA fragments containing m6A by using antibodies that specifically recognize m6A modification, and then determine the modified regions of m6A by high-throughput sequencing. But this technology has obvious defects:

• First, it depends on high-quality specific antibodies, and the batch difference and cross-reactivity of antibodies may lead to instability and false positive results.
• Second, the resolution is low, and it can only be located in the region of about 100-200nt, so it is impossible to achieve accurate detection of a single base level.
• Thirdly, it is difficult to make a quantitative analysis, which can not accurately reflect the abundance difference of the m6A modification.

These limitations make it difficult for researchers to accurately analyze the site-specific function and dynamic regulation mechanism of m6A modification. Therefore, it is urgent to develop a high-resolution, high-specificity, quantitative detection technique for m6A modification.

m6A dynamics in ADC tumors (Zeng et al., 2018)

Enzymatic Conversion-Based Detection in GLORI-seq

GLORI-seq technology takes enzymatic transformation as the core principle, and with the help of the specificity of human ALKBH5 demethylase, it realizes the efficient transformation of m6A to adenosine. By sequencing and comparing the modified sites, it provides an innovative scheme to break through the bottleneck of traditional technology and realize the mapping of m6A with single-base resolution.

Core Concept

GLORI-seq technology is a new detection technology of m6A modification, and its core concept is to remove m6A modification specifically and efficiently by using human ALKBH5 demethylase, so as to determine the site of m6A modification by detecting the base transformation in RNA sequence.

ALKBH5 is a demethylase dependent on Fe²⁺ and α-ketoglutarate, which can specifically recognize and catalyze the demethylation of adenosine modified by m6A and convert it into common A, but has no obvious effect on other RNA modifications, which ensures the high specificity of GLORI-seq technology.

Chemical Reaction Process

In the GLORI-seq technology, the key chemical reaction is the demethylation of m6A catalyzed by ALKBH5. Under suitable reaction conditions (including Fe, α-ketoglutaric acid, ascorbic acid, and other cofactors), ALKBH5 binds to an RNA chain containing m6A modification through its unique catalytic domain, and then removes the methyl group in m6A to convert m6A into adenosine. This chemical reaction has high efficiency and specificity, which can quickly and accurately transform the m6A modification site in RNA under in vitro conditions, laying a foundation for subsequent sequencing detection.

Principle of Sequencing Detection

GLORI-seq technology can identify the modified sites of m6A by high-throughput sequencing. Specifically, RNA samples treated with ALKBH5 and untreated control samples were sequenced, respectively. By comparing the sequencing results of the two samples, the site of base transformation can be determined. In untreated samples, the modified site of m6A still showed m6A during sequencing. However, in the sample treated by ALKBH5, m6A was converted into adenosine, so the site with different sequencing results in the two samples is the real m6A modification site. In this way, the GLORI-seq technology can accurately detect the m6A modification site.

Overview of m6A-mediated RNA metabolism (Meng et al., 2023)

The Workflow: From Library Preparation to Data Analysis

The experimental process of GLORI-seq is the core link to realize single-base resolution m6A detection, which runs through the whole process from RNA processing to data analysis. Based on the enzymatic transformation of ALKBH5, this process overcomes the limitations of traditional technology through rigorous library preparation and accurate bioinformatics analysis, and provides a standardized scheme for locating real m6A sites and quantifying the modification level, which is the key technical support for analyzing the dynamic regulation of the epigenome.

Details in Experimental Steps

• RNA fragmentation: First, high-quality total RNA or mRNA is extracted, and RNA is randomly fragmented into fragments of about 100-200nt by ultrasonic fragmentation or enzyme digestion for subsequent reaction and sequencing. The fragmentation process requires strict control of conditions to ensure the uniformity of fragment length and the integrity of RNA.
• In vitro demethylation reaction: The fragmented RNA was divided into two groups. One group was treated with ALKBH5 demethylase and the corresponding reaction buffer (including Fe, α-ketoglutarate, ascorbic acid, etc.), and incubated at a suitable temperature (usually 37℃) for a certain period of time to demethylate the m6A modification site. The other group, as the control group, did not add ALKBH5, but only added reaction buffer to incubate under the same conditions to eliminate the influence of non-specific reaction.
• Preparation of sequencing library: RNA fragments of the treatment group and the control group were prepared separately. Firstly, the RNA fragment was repaired at the end and treated with poly (A) tail. Then, a sequencing linker is connected, which contains a sample-specific index sequence for subsequent mixed sequencing and data analysis. Finally, the enrichment library was amplified by PCR, and a sufficient amount of DNA library was obtained for high-throughput sequencing.

Stoichiometric estimation with m6A modification ratio (Yu et al., 2024)

Bioinformatics Analysis Process

• Pre-processing of sequencing data: Quality control is carried out on the original data obtained by high-throughput sequencing, and low-quality reads, reads containing linker sequences, and repeated reads are removed to improve the reliability of the data. Commonly used quality control tools include FastQC and so on.
• Sequence alignment: Compare the sequenced reads of the pretreated treatment group and the control group to the reference genome by using the alignment software (such as HISAT2, STAR, etc.) to obtain the positioning information of the reads on the genome. Appropriate parameters need to be set in the process of comparison to ensure the accuracy and efficiency of the comparison.
• Identification of differential sites: By comparing the comparison results of the treatment group and the control group, the sites where the bases are transformed are identified. In the treatment group, because m6A is converted into adenosine, the site that was originally m6A in the control group will appear as A. However, the site where m6A modification did not occur was A in both groups. Therefore, through statistical analysis, it can be determined that the untransformed site (that is, the site where m6A is in the control group and m6A is still in the treatment group) is the m6A modification site. GLORI-seq technology can detect the resolution of a single base, accurate to the position of each nucleotide.
• Quantitative analysis: Based on the coverage of sequencing data and the ratio of base conversion, the abundance of m6A modification can be quantitatively analyzed. By calculating the difference in sequencing depth of specific sites between the treatment group and the control group, the level of m6A modification can be evaluated, which provides a quantitative basis for studying the dynamic changes of m6A modification.

Comparative Advantage of GLORI-seq

In the study of epigenomics, the performance of m6A modification detection technology is very important. GLORI-seq, miCLIP, and Mazter-seq are the mainstream technologies at present, but there are significant differences in their core characteristics. The following focuses on these three technologies and compares them from the dimensions of resolution, specificity, sample requirements, repeatability, and cost, revealing the unique advantages of GLORI-seq based on enzymatic transformation, and providing a reference for researchers to choose adaptive technologies.

Resolution Ratio

• GLORI-seq technology: It has single-base resolution and can determine the m6A modification site accurately to the position of each nucleotide. This is due to its detection principle based on enzymatic transformation. By directly comparing the base changes of the treatment group and the control group, a single m6A modification site can be accurately identified, which provides a powerful tool for studying the site-specific function of m6A modification.
• MiCLIP technology: This technology is based on ultraviolet cross-linking and immunoprecipitation, and determines the site of m6A modification by detecting cross-linking-induced mutation (such as C→T mutation). However, its resolution is limited by the distribution and detection sensitivity of cross-linking-induced mutations, and usually only reaches the single-base level. However, in practical application, it may be interfered with by background mutations, resulting in inaccurate positioning of some loci.

Generally speaking, GLORI-seq technology has obvious advantages in resolution, and it can draw the map modified by m6A more accurately.

Specificity

• GLORI-seq technology: Based on the enzymatic reaction, using the high specificity of ALKBH5 demethylase to m6A modification, it can only catalyze the demethylation reaction of m6A, but has no effect on other RNA modifications, so it has high specificity and can effectively reduce false positive results.
• MiCLIP technique: It belongs to the antibody-dependent immunoprecipitation method, and its specificity depends on the specificity of the anti-m6A antibody. However, antibodies may cross-react with other modifications with similar structures, leading to false positive results.
• Mazter-Seq technology: MazF can recognize and cleave a specific sequence (such as ACA) containing m6A modification based on the cleavage effect of restriction endonuclease MazF on the specific sequence. However, this enzyme is highly dependent on the sequence and may miss some m6A modification sites that do not contain its recognition sequence, so its specificity is relatively low.

Example of functional m6A methylation-related regulators in cancers (Li et al., 2023)

Input, Repeatability and Cost

• Sample input: GLORI-seq technology has low requirements for sample input, and usually only needs microgram RNA to carry out experiments, which is suitable for research with a limited sample size, such as clinical sample research. MiCLIP technology requires an immunoprecipitation step, which requires high sample input and usually requires milligrams of RNA. Mazter-Seq technology requires a sample input between the two.
• Repeatability: GLORI-seq technology is based on an enzymatic reaction; the reaction conditions are easy to control, and the influence of antibody batch difference is avoided, so it has high repeatability, and the results of different experimental batches are consistent. MiCLIP technology is greatly influenced by antibody quality and batch differences, and its repeatability is relatively low. The repeatability of Mazter-Seq technology is limited by the activity of the enzyme and the stability of reaction conditions.
• Cost: GLORI-seq technology does not need expensive, specific antibodies; the cost of the enzyme is relatively low, and the experimental process is relatively simple, so the overall cost is low, and it has high cost-effectiveness. MiCLIP technology needs to use high-quality, specific antibodies, and the cost is high, which increases the overall cost of the experiment. The cost of Mazter-Seq technology mainly depends on the price of the enzyme and the complexity of the experimental process, and the overall cost is between the two.

Structural insights into the YTH family proteins as m6A readers (Huang et al., 2018)

Conclusion

GLORI-seq technology, as a new detection technology of m6A modification, has shown great application potential in the study of apparent transcriptomics with its advantages of single-base resolution, high specificity, low sample input, high repeatability, and good cost-effectiveness, and is expected to become the gold standard quantitative method for mapping m6A modification. This technology realizes the accurate detection of m6A modification sites through enzymatic transformation, overcomes many limitations of the earlier technology, and provides a powerful tool for in-depth study of the biological function and dynamic regulation mechanism of m6A modification.

Using GLORI-seq technology, researchers can draw the map of m6A modification in different physiological and pathological states more accurately, find new m6A modification sites, and analyze the relationship between m6A modification and gene expression regulation, which provides a new perspective for understanding the mechanism of disease occurrence and development.

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