Analysis of mRNA 5'-terminal cap structures and internal N6-methyladenosine by reversed-phase high-performance liquid chromatography |
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Authors: | R J Albers B Coffin F M Rottman |
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Institution: | Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 USA |
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Abstract: | A simple procedure for determining the complete methylation profile of an mRNA molecule in a single chromatographic separation is described. The mRNA is selectively hydrolyzed to its component nucleosides leaving its cap 0 (m7GpppN′) or cap 1 (m7GpppN′m) structure intact. The hydrolysis products, which can include cap 0, cap 1, 2′-O-methylnucleosides (N″m) of cap 2 (m7GpppN′mpN″m) and internal N6-methyladenosine, are separated on an octadecyl reverse-phase column using a mobile phase containing acetonitrile and ammonium formate, a weak ion-pairing reagent. methyl-3H-labeled poly(A)-containing mRNA is used to demonstrate the efficacy of the procedure. |
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