Immunochemical detection of N6-methyladenine in DNA

Antibodies to N6-methyladenosine were produced in rabbit by means of immunization with N6-methyladenosine coupled to bovine serum albumin via periodate oxidation. Cross-reacting antibodies were removed by bovine serum albumin-Sepharose and appropriate nucleoside-human serum albumin absorbents. Nucleoside-specific antibodies were isolated by affinity chromatography of N6-methyladenosine-human serum albumin-Sepharose. The specificity of the purified antibodies has been demonstrated by complement fixation inhibition analyses using nucleoside analogues as inhibition than dAMP. Anti-N6-methyladenosine was used to detect N6-methyladenine in denatured DNAs from various sources by complement fixation. Practically no complement fixation has been found with DNAs containing no N6-methyladenine, such as calf thymus, salmon sperm, Micrococcus radiodurans, Streptomyces chrysomallus and Streptomyces hygroscopicus, whereas a weak reactivity occurred in the case of Bacillus subtilis DNA and Sarcina maxima DNA. For DNA from Proteus mirabilis, Escherichia coli K-12, E. coli B, E. coli WF+, lambda, T2 phages quantitative differences in the immunochemical reactivity were observed, which only partially correlate with the N6-methyladenine content of the DNAs. Other factors, influencing the accessibility of N6-methyladenine to the antibody-combining site have to be taken into consideration.