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. 2021 Nov 22;12:6777. doi: 10.1038/s41467-021-26926-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Tertiary-amine-containing ionizable lipids currently used in RNA-lipid nanoparticle (RNA-LNP) products. b N-oxide forms through tertiary amine oxidation and undergoes an acid/base-catalyzed hydrolysis at the amine to generate aldehydes and secondary amines. c N-oxide acid hydrolysis products were detected by reversed phase-ultra-high-performance liquid chromatography with charged aerosol detection and tandem mass spectrometry (RP-UPLC-CAD MS/MS); N-oxide standard (green), acid-precipitated N-oxide standard with (blue) and without (red) aminooxy-polyethylene glycol (aminooxy-PEG) label, and buffer baseline (black). Secondary amines from N-oxide hydrolysis elute at 8.7, 10.3, and 25 min. Corresponding aminooxy-PEG-derivatized aldehydes elute at 9.5 and 16 min, with the third likely in the column void. pA, pico ampere. d Binary reactions of mRNA with pure N-oxide result in high late eluting-peak (LP). RP-IP analysis of mRNA extracted from the binary is shown at 3 h (black), 1 day (blue), and 3 days (red) MP, mRNA main peak. e Binary reactions spiked with pure 17-carbon aldehyde and f pure 25-carbon aldehyde products from the N-oxide degradation pathway result in high levels of mRNA modification. Reversed phase-ion pair high performance liquid chromatography (RP-IP HPLC) chromatograms of the extracted mRNA are shown with no aldehyde spike (black), 0.5% aldehyde (red), and 2% aldehyde (green). g, h Binary reactions with the pure 17-carbon aldehyde from the N-oxide degradation pathway were analyzed for single nucleoside modifications. Binaries were prepared with no aldehyde (black), 0.5% aldehyde (red), 1% aldehyde (blue), and 2% aldehyde (green) spike. mRNA was extracted, enzyme-digested, and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The mass-to-charge ratios (m/z) corresponding to aldehyde-cytidine adducts (m/z 526.3 and 540.3) increased with aldehyde spike level. A representative ionizable lipid system, heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate is used here. Representative data from ≥ 3 repeat experiments are shown.