Extended Data Fig. 2: RNAse A assay analyzing decarboxylation of malonyl-tRNAs. | Nature Chemistry

Extended Data Fig. 2: RNAse A assay analyzing decarboxylation of malonyl-tRNAs.

From: Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo

Extended Data Fig. 2

a, Substituted malonic acid monomers used in this study. b, Left, 26a-d: malonyl adenosine nucleosides formed when malonyl-tRNA is digested by RNAse A; right, 27a and 27b: decarboxylation products of malonyl-adenosine nucleosides. c, LC-HRMS analysis of Ma-tRNAPyl acylation reactions after RNAse A digestion. Reactions were performed as described in Methods. The EIC for the malonyl product 26 as a mixture of 2’ and 3’ isomers (two pairs of diastereomers) (pink, top) shows the expected peaks whereas the EIC for the decarboxylation product 27, also as a mixture of 2’ and 3’ isomers (black, bottom) shows that the decarboxylation product is absent in all cases except the MaPylRS-catalyzed acylation of Ma-tRNAPyl with monomer 16. These data provide evidence that the decarboxylation of malonates charged to Ma-tRNAPyl observed in the intact tRNA mass analysis (Supplementary Figs. 7, 15, 17-19, 26, 28-30 and 32-35) occurs during either the workup or the LC-MS itself.

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