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Diastereoselectivity of 5-Methyluridine Osmylation Is Inverted inside an RNA Chain

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Institute of Pharmacy and Biochemistry, University of Mainz, D-55128 Mainz, Germany
*E-mail: [email protected]; tel: +49 6131 39 25731; fax: +49 6131 39 20373.
Cite this: Bioconjugate Chem. 2016, 27, 9, 2188–2197
Publication Date (Web):August 19, 2016
https://doi.org/10.1021/acs.bioconjchem.6b00403
Copyright © 2016 American Chemical Society

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    Abstract

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    In this study, we investigated the reaction of the osmium tetroxide–bipyridine complex with pyrimidines in RNA. This reagent, which reacts with the diastereotopic 5–6 double bond, thus leading to the formation of two diastereomers, was used in the past to label thymidine and 5-methylcytosine in DNA. In light of the growing interest in post-transcriptional RNA modifications, we addressed the question of whether this reagent could be used for labeling of the naturally occurring RNA modifications 5-methylcytosine and 5-methyluridine. On nucleoside level, 5-methylcytosine and 5-methyluridine revealed a 5- and 12-fold preference, respectively, over their nonmethylated equivalents. Performing the reaction on an RNA level, we could show that the steric environment of a pentanucleotide has a major detrimental impact on the reaction rate of osmylation. Interestingly, this drop in reactivity was due to a dramatic change in diastereoselectivity, which in turn resulted from impediment of the preferred attack via the si side. Thus, while on the nucleoside level, the absolute configuration of the major product of osmylation of 5-methyluridine was (5R,6S)-5-methyluridine glycol-dioxoosmium-bipyridine, reaction with an RNA pentanucleotide afforded the corresponding (5S,6R)-diastereomer as the major product. The change in diastereoselectivity lead to an almost complete loss of selectivity toward 5-methylcytosine in a pentanucleotide context, while 5-methyluridine remained about 8 times more reactive than the canonical pyrimidines. On the basis of these findings, we evaluate the usefulness of osmium tetroxide–bipyridine as a potential label for the 5-methyluridine modification in transcriptome-wide studies.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00403.

    • Figures showing NMR spectra and other analysis results, the linear dependency of the natural logarithm of normalized concentration over time, and normalized uridine decay in a time-dependent manner. Tables containing NMR comparison analysis and quantification analysis and a summary of reaction rate constants. Additional details on NMR analysis. (PDF)

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    Cited By

    This article is cited by 7 publications.

    1. Mark Helm, Martina C. Schmidt‐Dengler, Marlies Weber, Yuri Motorin. General Principles for the Detection of Modified Nucleotides in RNA by Specific Reagents. Advanced Biology 2021, 5 (10) https://doi.org/10.1002/adbi.202100866
    2. Turja K Debnath, Blerta Xhemalçe. Deciphering RNA modifications at base resolution: from chemistry to biology. Briefings in Functional Genomics 2021, 20 (2) , 77-85. https://doi.org/10.1093/bfgp/elaa024
    3. Catherina Gasser, Isabel Delazer, Eva Neuner, Katharina Pascher, Karl Brillet, Sarah Klotz, Lukas Trixl, Maximilian Himmelstoß, Eric Ennifar, Dietmar Rieder, Alexandra Lusser, Ronald Micura. Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL). Angewandte Chemie 2020, 132 (17) , 6948-6953. https://doi.org/10.1002/ange.201916272
    4. Catherina Gasser, Isabel Delazer, Eva Neuner, Katharina Pascher, Karl Brillet, Sarah Klotz, Lukas Trixl, Maximilian Himmelstoß, Eric Ennifar, Dietmar Rieder, Alexandra Lusser, Ronald Micura. Thioguanosine Conversion Enables mRNA‐Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC‐seq DUAL). Angewandte Chemie International Edition 2020, 59 (17) , 6881-6886. https://doi.org/10.1002/anie.201916272
    5. Madiha Sultan, Anastassia Kanavarioti. Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag. Scientific Reports 2019, 9 (1) https://doi.org/10.1038/s41598-019-50459-8
    6. Turja Kanti Debnath, Akimitsu Okamoto. Osmium Tag for Post‐transcriptionally Modified RNA. ChemBioChem 2018, 19 (15) , 1653-1656. https://doi.org/10.1002/cbic.201800274
    7. Matthias Heiss, Stefanie Kellner. Detection of nucleic acid modifications by chemical reagents. RNA Biology 2017, 14 (9) , 1166-1174. https://doi.org/10.1080/15476286.2016.1261788

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