High-Throughput Spectroscopic Analysis of mRNA Capping Level

Eukaryotic mRNAs are characterized by terminal 5′ cap structures and 3′ polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a novel biosensor method designed to detect the presence of both cap structures and polyadenylation sites on mRNA molecules. This novel biosensor is sensitive to mRNA degradation and can quantitatively determine capping levels of mRNA molecules within a mixture of capped and uncapped mRNA molecules. The biosensor displays a constant dynamic range between 254 nt and 6507 nt with reproducible sensitivity to increases in capping level of at least 20% and a limit of detection of 2.4 pmol of mRNA. Overall, the biosensor can provide key information about mRNA quality before mammalian cell transfection.