Development and validation of monoclonal antibodies against N6-methyladenosine for the detection of RNA modifications

Abstract

RNA contains various chemical modifications, among which N6-methyladenosine (m6A) is the most prevalent modified nucleotide in eukaryotic mRNA. Emerging evidence suggests that m6A plays an important role in regulating a variety of cellular functions by controlling mRNA processing, translation and degradation. Because m6A is not detectable by standard chemical modification-based approaches, immunological methods, such as ELISA, immunoblotting, immunohistochemistry, m6A RNA immunoprecipitation sequencing and m6A individual-nucleotide resolution cross-linking and immunoprecipitation, have been employed to detect m6A in RNA. Although the most important factor determining the success of these methods is the integrity of highly specific antibodies against m6A, the development of m6A-specific monoclonal antibodies has been challenging. We developed anti-m6A monoclonal antibodies using our recently developed single cell-based monoclonal antibody production system. The binding of one selected antibody, #B1-3, to RNA oligoribonucleotide containing a single m6A had an equilibrium dissociation constant of 6.5 nM, and this antibody exhibited negligible binding to oligoribonucleotides containing a single N1-methyladenosine and unmodified adenosine. The binding was competed by the addition of increasing concentrations of N6-methyl-ATP but not N1-methyl-ATP or ATP. Furthermore, this mAb specifically crosslinked m6A-containing oligoribonucleotide by ultraviolet light, resulting in the induction of cDNA truncation at m6A position. These results show the feasibility of using the validated m6A monoclonal antibody for the specific detection of m6A in RNA.

Direct Immunodetection of Global A‐to‐I RNA Editing Activity with a Chemiluminescent Bioassay

“…[49,50] These qualities have positioned ELI-SA as aroutine research technique and apowerful diagnostic tool, and have enabled sensitive detection of human antibody titers, [51] cytokine and small-molecule analytes in serum, [52] and viral particles in different biological fluids. [53] Moreover, primary antibodies identified for other rare nucleic acid modifications,i ncluding N 6 -methyladenosine (m 6 A), [54,55] 5methylcytosine (5mC), [56] 5-hydroxymethylcytosine (5hmC), and 5-carboxylcytosine (5caC), [57] have now also been successfully deployed in commercial ELISA formats for sensitively detecting global editing rates in RNAa nd DNA. ELISA also utilizes inexpensive consumables and commonplace equipment.…”

Fat mass and obesity‐associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

“…Although m 6 A methylation was discovered in 1974 (Desrosiers et al, 1974), few studies were performed on the biological functions of m 6 A modification due to limited detection strategies. Detection on m 6 A level is challenging because m 6 A is not detectable by traditional chemical approaches or immunological methods, such as enzyme‐linked immunosorbent assay and immunoblotting (Matsuzawa et al, 2019). In 2015, m 6 A‐specific MeRIP approach was employed to probe the m 6 A peak site in fragmented mRNA with a ∼100‐nucleotide resolution using m 6 A‐specific antibodies (K. Chen et al, 2015).…”

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

“…The use of specific antibodies for the detection of RNA modifications was proposed and successfully implemented in the late 1970s [ 108 , 109 , 110 ], and numerous polyclonal and monoclonal antibodies were used for analysis of DNA- and RNA-modified nucleotides (as reviewed in [ 111 ]). This development is still ongoing and highly specific antibodies can be obtained in this manner (see [ 112 ]). However, the majority of antibodies against modified nucleotides/nucleosides have a poor affinity and specificity [ 113 ] and, therefore, enrichment factors for modified RNA are only very modest [ 114 ].…”