Validating tRNA Integrity: CCA-3' Ends

The integrity of the single-stranded 3′-CCA ends of the in vitro transcribed tRNAs was tested with a fluorescently labeled RNA/DNA stem loop oligonucleotide (Supplementary Table 3) ligated to the tRNA with 2.5 U T4 DNA ligase (Thermo Fisher Scientific) in 5 µL T4 DNA ligase buffer with 15% (v/v) DMSO overnight at 16 °C. tRNAs with ligated hairpin oligonucleotide and intact CCA termini were separated from the bulk tRNAs on denaturing PAGE. RNAs were visualized by fluorescence or SYBR^TM Gold Nucleic Acid Stain. The approach visualizes only tRNAs with intact CCA-3′ ends and isoacceptors with aberrantly in vitro synthesized 3′ ends or truncated CCA ends in vivo remain invisible^{61 (link)}.