RNase H-based analysis of synthetic mRNA 5′ cap incorporation

Uniform RNase H cleavage with designed targeting oligos. (A) 5′ sequence of a 1.7 kb in vitro FLuc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. (B) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.