Characterization of antibodies specific for N⁶-methyladenosine and for 7-methylguanosine

Abstract

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were prepared by immunization of rabbits with nucleoside conjugates of bovine serum albumin (i.e, m6A--BSA and m7G-BSA). Specificity of each antibody was assessed by inhibition of the homologous precipitin reaction with various nucleosides. These analyses revealed that the antibodies elicited in response to m6A--BSA were specific for the N6-methyl moiety of adenosine with minimal or no cross-reactivity with BSA, adenosine, and guanosine. Although a major fraction of antibodies elicited in response to m7G--BSA were specific for m7G, considerable cross-reactivity was observed with BSA. These latter antibodies were removed by affinity chromatography utilizing BSA-Sepharose adsorbent. In similar fashion, antibodies specific for m6A and m7G were isolated by immunospecific adsorption to antigen-coupled Sepharose (e.g. m6A--BSA-Sepharose), eluted, and coupled to Sepharose. The ability of these antibody-coupled adsorbents to retain specific methylated [methyl-3H]nucleosides derived from [methyl-3H]tRNA digests was assessed. Both the anti-m7G and anti-m6A antibody adsorbents quantitatively and exclusively retained 7-[3H]methylguanosine and N6-[3H]methyladenosine, respectively. The application of these adsorbents to fractionate oligonucleotides and nucleic acids is discussed.

Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons

“…For these experiments, we used a previously described anti-m 6 A antibody (Bringmann and Luhrmann, 1987; Jia et al, 2011; Munns et al, 1977). To ensure the specificity of this antibody for m 6 A, we performed dot blots using modified oligonucleotides immobilized to a membrane.…”

RNA m6A methylation regulates the ultraviolet-induced DNA damage response

“…As the m 6 A antibody recognizes both modified DNA and RNA 10 , we investigated which type of nucleic acid was modified following UV irradiation. RNase A treatment of cells abrogated m 6 A accumulation at damage sites (Extended Data Fig.…”

Deep Sequencing Shows Multiple Oligouridylations Are Required for 3′ to 5′ Degradation of Histone mRNAs on Polyribosomes

“…We sequenced capped histone mRNAs isolated by immunoprecipitation with anti-m 7 G with an anti-m 7 G antibody (Munns et al, 1977) from cells treated with HU for 20 min. Most of the histone mRNA and GAPDH mRNA were precipitated, as determined by RT-PCR (Fig.…”