A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′-O-methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).
