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

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.