Extended Data Fig. 4: Chemical structure determination of N³²⁴.

(a) RNA-MS of the N³²⁴-containing fragment of tRNA^Val3 digested with RNase T₁, with (+) or without (-) BAP treatment. XICs show the divalent negative ions of N³²⁴m⁵Cm⁵CUGp (m/z 845.59, z = -2) and Um⁵Cm⁵CUG-_OH (m/z 765.52, z = -2). Two phosphates were removed by this treatment. (b) CID spectrum of the N³²⁴-containing fragment treated with BAP. The product ions are assigned on Um⁵Cm⁵CUG-_OH. (c) Preparation scheme of pN³²⁴p. S. tokodaii tRNA fraction is digested with nuclease P₁, yielding dinucleotide pN³²⁴m⁵C. The digests were subjected to periodate oxidation and β-elimination to remove the 3′ terminal residue. The resultant pN³²⁴p was purified by anion exchange chromatography and subjected to LC/MS/MS analysis. (d) LC/MS nucleotide analysis of the nuclease P₁ digest of S. tokodaii tRNA fraction. UV trace at 254 nm (upper panel) and XIC of the negatively charged ion of pN³²⁴m⁵C (m/z 722, z = -1) (lower panel) are shown. (e) CID spectrum of pN³²⁴m⁵C. The product ions were assigned on the predicted chemical structure of pN³²⁴m⁵C. The phosphate group of N³²⁴ is shown in red. (f) CID spectrum of the N³²⁴ nucleotide (pN³²⁴p; m/z 483, z = -1). The product ions are assigned in the predicted chemical structure of pN³²⁴p. (g) RNA-MS of the V-loop-containing RNA fragment with (+) or without (-) Tpt1p treatment before RNase T₁ digestion. XICs show the divalent negative ions of U^pm⁵Cm⁵CUGp (m/z 845.59, z = -2) and Um⁵Cm⁵CUGp (m/z 805.60, z = -2). (h) CID spectrum of the dephosphorylated fragment by Tpt1p. The Tpt1p-treated tRNA^Val3 was digested with RNase T₁ and analyzed by RNA-MS. The V-loop containing fragment was selected as a precursor for CID. The product ions are assigned on the sequence as indicated.