Quantification of tRNA m 1 A modification by templated-ligation qPCR.

N1-methyladenosine (m 1 A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m 1 A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m 1 A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m 1 A, a PCR method to assess m 1 A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m 1 A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m 1 A interferes with the ligation in specific ways, allowing for the quantitative assessment of m 1 A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m 1 A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.