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bioRxiv · 10.64898/2026.05.23.727428

RNase 4 improves bottom-up modification mapping of E. coli total tRNAs using HILIC-MS/MS

Simcox, K. M.·Zamecnik, M.·Kennedy, R. T.·Koutmou, K. S.
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Abstract

The structural and functional diversity of RNAs is expanded by the post-transcriptional incorporation of nucleoside variants. Emblematic of this, tRNAs contain extensive modifications that ensure their function during protein synthesis. Mass spectrometry has long been the field standard for identifying specific sites of chemical modifications on RNA. Nonetheless, mass spectrometry-based mapping approaches are not widely implemented. This is partially due to technical challenges associated with current methodologies including the limited diversity of available RNases, complexity of RNA mixtures, and conventional use ion-pairing reagents that require dedicated instrumentation. Here, we present a bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow employing hydrophilic interaction liquid chromatography (HILIC) without ion-pairing reagents to globally map E. coli tRNA modifications. We implement orthogonal digestions using RNase 4 and a folded digestion scheme with RNase T1 to generate uniquely mappable oligonucleotides compatible with HILIC-MS/MS analysis and achieve 75-100% sequence coverage for most tRNA isoacceptors. HILIC-MS/MS matches the performance of traditional ion-pairing reverse-phased LC-MS/MS. This level of coverage allowed us to discover a new site of methylation (Gm17) in tRNAGly, and confirm the presence of an s4U8 modification predicted in tRNAArg. Furthermore, by applying this method to E. coli lacking the m5U54 methyltransferase (trmA) we confirmed the established dependence of acp3U47 insertion on m5U54 in tRNAPhe. Our findings show that RNase 4 improves bottom-up tRNA sequencing, enabling high-quality E. coli tRNA analysis without ion-pairing reagents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/727428v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1f483a3org.highwire.dtl.DTLVardef@1ee5e01org.highwire.dtl.DTLVardef@5db88borg.highwire.dtl.DTLVardef@fee855_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Simcox, K. M., Zamecnik, M., Kennedy, R. T., Koutmou, K. S.. 2026-05-25. RNase 4 improves bottom-up modification mapping of E. coli total tRNAs using HILIC-MS/MS. https://doi.org/10.64898/2026.05.23.727428

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