Antibodies distinguishing between intact and alkali-hydrolyzed 7-methylguanosine

Abstract

Antibodies specific for intact 7-methylguanosine (m 7 G) were induced in rabbits and mice by immunization with nucleoside-BSA or nucleoside-hemocyanin conjugates. Since m 7 G undergoes alkali-catalyzed hydrolytic fission of the purine ring, modifications were made in the procedure for conjugation of m 7 G to proteins. After periodate oxidation, m 7 G was incubated with protein at pH 9.1 at 4°C for one hour during which the nucleoside was found to be stable. Reduction of the Schiff base was done with t-butylamine borane for 30 minutes, and the conjugated protein was isolated quickly by gel filtration at pH 7.2. Both rabbits and mice produced antibodies that readily distinguished between the intact and hydrolyzed m 7 G. Antibody specificity depended largely on the presence of an intact 7-substituted imidazole ring and some cross-reaction occurred with 7-methylinosine. A weaker reaction occurred with ribothymidine and thyinidine. Mouse antibodies induced by m 7 G-hemocyanin showed the highest specificity. They also recognized m 7 G in the isolated mRNA cap structure m 7 G(5′)ppp(5′)A.