Quantification of tRNA m¹A modification by templated-ligation qPCR

TL-qPCR on human tRNA_i^Met mimicking RNA oligonucleotides. (A) The oligos and linkers used in SplintR ligation for tRNA_i^Met-m¹A58. A oligo and m¹A oligo: two RNA oligos containing A or m¹A-modified nucleotide (indicated with ″ according to conventional RNA modification nomenclature) as underlined. PF and PR: primer-binding site for PCR. HEX: qPCR fluorescent probe binding site. (B) qPCR curves and (C) relative PCR product amounts of the four linkers 5lkrT/C/G/A in the templated SplintR ligation using 0.1 nM A oligo as input. (D) Amplification curves and (E) standard ΔCq curve of the dilution series of the A oligo input (#1 to #7 for 10× serial dilution from 1 nM to 1 fM) with H₂O as a negative control (#8), generated with the linker 5lkrT in the SplintR ligation. (F) Standard Cq curves of 1 nM total m¹A/A oligo mixture inputs in the SplintR ligation using the linker 5lkrT.