Dot blot (or slot blot) technology is used in molecular biology as a semiquantitative or quantitative detection method for DNA, RNA and protein samples, and is predominantly used semiquantitatively in m⁶A analysis (32,33,55-57). Compared with western blotting, northern blotting or Southern blotting methods, dot blot technology has a simpler operation with a similar working principle. The biomolecules are not subjected to electrophoretic separation prior to detection with the dot blot method. Instead, samples containing a mixture of RNAs are directly applied to a membrane through an apparatus with circular templates that form a dot when the sample is applied. After applying a vacuum to embed RNAs and dry the membrane, biomolecules are detected with antibodies. When detecting RNA, the DNA must be removed prior to loading to eliminate its influence on m⁶A (32,33). A schematic diagram of the determination of m⁶A modification residues by dot blot is presented in Fig. 2.

Dot blot technology markedly saves time, since it does not require chromatography, gel electrophoresis or complex gel blocking procedures (32,33). However, regarding m⁶A detection, dot blot technology is only able to verify the presence of m⁶A or compare the amounts of m⁶A between different groups (32,33). Li et al (32) improved the traditional dot blot method to measure the global m⁶A abundance in the transcriptomes of four acute myeloid leukemia cell lines. However, this method is still not able to quantitate or precisely determine the location of m⁶A.

The regulatory enzymes of m⁶A have been reported to contribute to tumorigenesis (58-60). While the significance of m⁶A has been confirmed, no convenient approach has been developed to analyze m⁶A methyltransferase and demethylase activities or to monitor the inhibitors of these activities.

The Escherichia coli toxin MazF exhibits endoribonuclease activity specific against ACA sequences and is susceptible to m⁶A. MazF is the first enzyme discovered with the ability to specifically cleave RNA containing m⁶A, and is used to study activities of m⁶A demethylase and methyltransferase (34). Furthermore, MazF has an application for monitoring the inhibitors of m⁶A methyltransferase and demethylase. RNA cleavage by MazF may be detected by polyacrylamide gel electrophoresis and the fluorescence-resonance energy transfer-based plate assay (61). A schematic diagram of the determination of m⁶A modification residues by methyl-sensitive MazF RNA endonucleases is presented in Fig. 3.

At the current stage of development, the MazF cleavage methods are restricted regarding the evaluation of m⁶A, as MazF is only able to cleave the 5′-ACA-3′ site in single-stranded RNA, frequently occurring in endogenous RNAs. However, MazF is not able to cleave the 5′-ACA-3′ site in double-stranded RNA (61), and thus, it may not accurately determine the presence of m⁶A in structured RNA.

The immuno-northern blot combines a northern blotting experimental program and m⁶A-binding antibody, which is different from conventional northern blot techniques that use DNA probes. Immuno-northern blot does not require RNA fragmentation prior to analysis and the RNAs are separated based on their molecular weights (35). Thus, the detection of m⁶A modifications by immuno-northern blot is applied in various types of RNA (35).

In brief, RNAs are separated in a denaturing acryl-amide gel or an agarose gel and then transferred onto nylon membranes. The RNA strands on the membranes are exposed to ultraviolet (UV) irradiation for cross-linking, followed with incubation with primary antibodies against m⁶A modifications, corresponding secondary antibody and chemiluminescent detection (35).

The regulatory role of mRNA predominantly depends on the interaction between mRNA and RNA-binding proteins to regulate RNA splicing, stability, localization and translation (62). Photo-crosslinking technologies are diffusely applied to stabilize direct protein-RNA interactions (63). These technologies depend on the tendency for UV-induced photochemistry of nucleobases, which are natural or derivatives containing sulfur or halogen substituents. Photo-affinity labels, including diazirine (64) or benzophenone (65), are not widely used in the analysis of protein-RNA interactions; however, they may be activated by longer wavelengths and provide more efficient crosslinking.

Arguello et al (29) developed a chemical proteomics approach based on photo-crosslinking of the RNA base and diazirine, which was highly efficient in quantitatively analyzing protein-RNA interactions regulated by m⁶A modification. By using this method, novel m⁶A 'readers' have been discovered. To isolate m⁶A readers with photosynthetic and quantitative proteomics, RNA probes are required to contain the following: i) m⁶A molecules; ii) a photo-crosslinker that is efficient and does not influence the protein-RNA interactions; iii) streptavidin as an affinity handle for protein enrichment. A probe was prepared that contains the sequence GGm⁶ACU, the common recognition pattern of the m⁶A site in mammalian cells. This sequence is indispensable for binding the YTH-domain proteins (66,67). The probe was validated by known m⁶A RNA 'readers', including YTHDF1 (20), YTHDF2 (21), YTHDF3 (3,22,68) and YTHDC1 (22). A schematic diagram of the detection of m⁶A modification residues by the RNA photo-crosslinkers and quantitative proteomics technologies is presented in Fig. 4. We hypothesize that the requirement of the synthesis of the sensor may be a limitation regarding these techniques.

The majority of the abovementioned analytic methods are difficult to perform and expensive. The electrochemical immunosensor method was developed to provide convenience and high sensitivity (30). An anti-m⁶A antibody has been used to detect m⁶A by targeting m⁶A-5′-triphosphate (m⁶ATP). The detection and capture of m⁶A relies on silver nanoparticles and SiO₂ (Ag@SiO₂) nano-spheres with amine-polyethylene glycol 3-biotin. Ag@SiO₂ nanospheres were prepared to amplify signals. Phos-tag-biotin was prepared to link m⁶ATP and Ag@SiO₂ through two types of specific interaction between phosphate group of m⁶ATP and phos-tag, biotin and streptavidin, respectively. Experiments for evaluating this strategy indicated that the immuno-sensor has acceptable reproducibility and specificity with a wide linear range and a low detection limit. A schematic diagram for the determination of m⁶A modification residues by the electrochemical immunosensor method is presented in Fig. 5.

The efficacy of the detection of the m⁶A content using the electrochemical immunosensor method had been verified in human cell lines (30). It also provides a technological basis for the detection of RNAs and DNAs with the advantages of convenience, low cost, and high specificity and sensitivity.

High-throughput next-generation sequencing-based technology for identifying m⁶A sites based on where adenosine is methylated has not been applied in most species. In recent years, a vector machine method was developed to identify m⁶A sites in Arabidopsis thaliana (37). When combining anti-m⁶A antibodies and high-throughput sequencing, Luo et al (69) obtained thousands of m⁶A peaks for A. thaliana, including 'common' m⁶A peaks. Since the RRACH motif, where R resembles purine, A stands for m⁶A and H resembles a non-guanine base (69), was identified in most of the m⁶A peaks, Chen et al (37) collected segments containing RRACH at the center of the 'common' m⁶A peaks and proposed a model that may accurately identify specific m⁶A sites with high accuracy.

If the model is adapted to other plant species other than A. thaliana, this vector machine-based method may be used for the detection of m⁶A and other post-transcriptional modifications in these other plants as well.

HRM analysis is a simple method to detect m⁶A modification residues at a specific location in RNAs (38). HRM may be applied to high-throughput measurement. The resulting HRM curves of the samples of RNA mixtures change steadily from 100% of methylated RNA to 100% of unmethylated RNA (38). As presented in Fig. 6, the detection of m⁶A modification residues by the HRM method relies on the modified nucleoside position at a particular site of RNA and is followed by rapid screening for conditions or genes necessary for analysis of that modification (38).

According to the specificity of the oligonucleotide probe hybridization, bulk cellular RNA, as opposed to purified specific RNA, has been designed for detecting m⁶A at a pre-defined position (38). In addition, partial non-ribosomal target enrichments may be easily accomplished using commercially available kits.

A possible application for this method would be to screen knockout/knockdown strain libraries to identify genes contributing to the formation of a specific m⁶A nucleoside. Another possible application is to detect the presence of a particular m⁶A nucleoside under different growth or environmental conditions (2,3). HRM analysis may help to elucidate the dynamic events that result in the modification of certain RNAs.

For thorough investigation of the m⁶A epitranscriptome, Molinie et al (27) invented the m⁶A-LAIC-seq technique. Combined with RNA IP whole-transcriptome sequencing, m⁶A-LAIC-seq may quantify m⁶A contents, with spike-in RNAs as an internal standard. A schematic diagram for the determination of m⁶A modification residues by m⁶A-LAIC-seq is presented in Fig. 7. The results demonstrate a quantitative road map to which genes are the most or the least likely to be influenced by m⁶A-dependent regulatory networks. This method may determine the m⁶A levels in each gene but cannot stoichiometrically analyze the methylation of a single modified nucleotide. m⁶A-LAIC-seq complements m⁶A-seq identification of methylation sites and helps to expand the understanding of the biology of m⁶A.

To elucidate the dynamic biological functions of m⁶A, Liu et al (40) provided the SCARLET method that directly measures the precise location and the status of m⁶A modification at any candidate site of mRNA/lncRNA at a single nucleotide resolution. In addition to m⁶A, SCARLET may be used to observe other RNA modifications, including 5-methylcytosine, pseudouridine and 2′-O-methyl ribonucleosides. SCARLET is available to study the biological functions of RNA modifications with general experimental equipment and materials. A schematic diagram for the determination of m⁶A modification residues by SCARLET is presented in Fig. 8.

The feasibility of using the SCARLET method has been confirmed in HeLa cell RNA samples, which produced similar results compared with previously reported m⁶A sites in Hela samples. Using this method, a minimally modified m⁶A site that was not precisely determined in a preceding study was also identified, demonstrating that SCARLET is able to easily resolve the ambiguity of modification sites (70).

Although RNA methylation has been identified and verified in the 1970s, the relevant modification mechanism, regulatory means and biological significance have not been clarified due to technical limitations. The recent emergence of MeRIP-Seq technology makes it possible to study m⁶A methylation at the transcriptome level by high-throughput sequencing (2,3).

MeRIP-Seq is a combination of ChIP-Seq and RNA-Seq that is able to elucidate global mRNA m⁶A sites in mammalian cells. MeRIP-seq is a novel type of IP-seq technology, in which the known ChIP-seq, photoactivatable ribonucleo-side-enhanced crosslinking and IP (a more mature sequence), is applied. MeRIP-seq has been successfully used to detect whole-genome m⁶A modifications (42-46). The major principle of this technique is as follows: The anti-m⁶A antibody is incubated with randomly interrupted RNA fragments using a co-IP method resulting in an m⁶A modification fragment that is precipitated and sequenced. Concurrently, a control sample is run that eliminates the background during antibody capture (2).

Successful preparations of each library should be evaluated prior to massive parallel sequencing. To identify and localize the m⁶A sites at a transcriptome-wide level by m⁶A IP, fragmentation of poly(A)⁺-selected RNAs (input) is required prior to IP with anti-m⁶A antibodies. The input and m⁶A IP RNAs are then separately processed for next-generation sequencing (2). Zhang et al (48) first proposed a Bayesian statistical model, BaySeqPeak, to analyze MeRIP-Seq data to help discover methylation site signals in the transcriptome. A reference transcriptome was prepared, which contains a single, non-intron splice variant of each gene. The resultant reads were then specifically compared with the reference to identify m⁶A sites with a low false-detection rate (3). A schematic diagram for the determination of m⁶A modification residues by MeRIP-Seq is presented in Fig. 9.

It has been reported that m⁶A residues may be located by producing unique signature mutations with anti-m⁶A antibodies and UV crosslinking techniques (31). m⁶A residues were mapped with two antibodies, one of which translates C to T to detect single and clustered m⁶A residues, and the other antibody that produces truncations is used to determine the position of m⁶A sites and detect m⁶A residues concurrently. A schematic diagram for the determination of m⁶A modification residues by miClip is provided in Fig. 10.

Identification of m⁶A residues by direct detection is superior to that by bioinformatics predictions from MeRIP-Seq peaks (31). The reliability of bioinformatics prediction depends on the characteristics of the m⁶A peak. MeRIP-Seq can accurately predict m⁶A residues only with a single clear peak of a single m⁶A residue and is limited to the centrally-located DRACH motif (31). m⁶A residues usually cluster in mRNAs, resulting in multiple MeRIP-Seq peaks (2). Identification of m⁶A residues using miCLIP technology is not influenced by peak shapes and not confined to the centrally-located DRACH motif. Therefore, miCLIP can correctly identify m⁶A residues.

At present, RNA samples are prepared by antibody-based enrichment of m⁶A residues prior to sequencing, as m⁶A modifications are usually lost after reverse transcription (25,26). The indirect detection may lead to a higher error rate, pushing the generation of novel DNA polymerase to sequence m⁶A directly (26). In this light, Aschenbrenner et al (26) developed a screening method to develop a reverse transcription-active KlenTaq DNA polymerase variant for labeling N⁶-methylation residues. A schematic diagram for the determination of m⁶A modification residues by the DNA polymerase for direct N⁶-methyladenosine sequencing is presented in Fig. 11.

The development of HPLC occurred only 30 years ago; however, the development of this separation analysis technology is very rapid, and is now widely used, including in the detection of m⁶A modification (50). HPLC was firstly used to detect m⁶A modification in nine DNAs (50). Subsequently, Rana and Tuck (49) applied HPLC in the detection of m⁶A modification in a T7 RNA transcript coding for mouse dihydrofolate reductase by separating m⁶A from adenine, cytosine, uracil and so on. In a study by Jia et al (18), HPLC helped observe the changes of the m⁶A ratio in mRNAs following FTO treatment to better understand the function of FTO. The development of this in vitro methylation assay opened the door for studies investigating m⁶A levels in specific mRNAs or learn the biological significance of m⁶A modification.