Fig. 1: Acylation-dependent tRNA extension enables the sensitive detection and isolation of acylated tRNAs.

a, Schematic of fluoro-tREX and bio–tREX protocols. tRNAs are isolated from cells, and the diol functionality of the 3′ ribose on non-acylated tRNAs is oxidized to the dialdehyde. The acyl group of charged tRNAs protects the diol functionality of the 3′ ribose and prevents oxidation to a dialdehyde. A Cy3-labelled DNA probe complementary to the 3′ end of a target tRNA is annealed, and target tRNAs that were acylated are extended upon addition of Klenow fragment exo− and modified nucleotides. For fluoro-tREX, Cy5-labelled nucleotides are incorporated. Acylated tRNAs lead to a Cy5 and Cy3 signal, whereas non-acylated tRNAs only give a Cy3 signal. For bio–tREX, biotinylated nucleotides are incorporated and the tRNAs that were acylated are purified using streptavidin beads and can be visualized by SYBR gold staining following gel electrophoresis. b, Fluoro-tREX detected the acylation of \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) in the presence of PylRS and BocK (1). Cells expressing \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) were grown in the presence and absence of PylRS and BocK (1). c, Bio–tREX enables the selective isolation of previously acylated tRNAs. Cells harbouring \({{\rm{tRNA}}}_{{\rm{C}}{\rm{U}}{\rm{A}}}^{{\rm{P}}{\rm{y}}{\rm{l}}}\) were grown in the presence and absence of PylRS and BocK (1). Isolation of the tRNA and associated probe was visualized by SYBR Gold staining for RNA and Cy3 fluorescence for the probe. Experiments in b,c were repeated three times with similar results. For full, uncropped gels for all figures see Supplementary Fig. 1.