Analysis of tRNA Aminoacylation by Northern Blotting and Multiplex Small RNA-Seq

Measuring RNA folding is particularly challenging for tRNAs, where extensive structure and posttranscriptional modifications complicate traditional structure probing and reverse-transcriptase-based assays. Since tRNA folding is a prerequisite for aminoacylation, quantifying tRNA aminoacylation is often used as an indirect, though functionally relevant, measure of tRNA folding. Periodate oxidation and beta-elimination, resulting in the selective loss of the 3' terminal nucleotide of deacylated tRNAs, is an amino-acid-agnostic chemistry that enables differentiation of aminoacylated and deacylated tRNAs when coupled to high-resolution northern blotting or RNA sequencing. Quantification of tRNA aminoacylation has been used to demonstrate RNA chaperone activity for RNA-binding proteins that promote tRNA folding to increase the fraction of aminoacylated tRNAs, underscoring the utility of this assay in interrogating RNA chaperone-assisted tRNA folding and function.