Quantification of tRNA m¹A modification by templated-ligation qPCR

N1-methyladenosine (m¹A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m¹A 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 m¹A 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 m¹A, a PCR method to assess m¹A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m¹A 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. m¹A interferes with the ligation in specific ways, allowing for the quantitative assessment of m¹A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m¹A-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.