Total Synthesis of the Hypermodified RNA Bases Wybutosine and Hydroxywybutosine and Their Quantification Together with Other Modified RNA Bases in Plant Materials

Hienzsch, Antje; Deiml, Christian; Reiter, Veronika; Carell, Thomas

doi:10.1002/chem.201204209

Cited by 16 publications

(14 citation statements)

References 33 publications

(45 reference statements)

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“…There are commercial sources for a few isotopically labeled ribonucleosides and several research groups have synthesized specific standards (Brandmayr et al, 2012; Hienzsch, Deiml, Reiter, & Carell, 2013). To overcome this limitation, Helm and coworkers developed a biosynthetic approach that is accessible to any laboratory equipped for LC-MS work.…”

Section: Methodsmentioning

confidence: 99%

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Cai

Chionh

Hia

et al. 2015

Methods in Enzymology

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Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. The chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: 1) RNA isolation, 2) RNA purification, 3) RNA hydrolysis to individual ribonucleosides, 4) chromatographic resolution of ribonucleosides, 5) identification of the full set of modified ribonucleosides, 6) mass spectrometric quantification of ribonucleosides, 6) interrogation of ribonucleoside datasets, and 7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.

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Section: Methodsmentioning

confidence: 99%

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Cai

Chionh

Hia

et al. 2015

Methods in Enzymology

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“…In weiteren Arbeiten wurden beispielsweise die hypermodifizierten Basen Wybutosin (49) und Hydroxywybutosin (50) synthetisiert. Sie dienten einer Quantifizierung dieser Spezies in Pflanzen 75. Ebenso wurde das hypermodifizierte tRNA‐Nukleosid Epoxyqueuosin (52) synthetisiert und damit dessen Struktur verifiziert 76.…”

Section: Oligonukleotideunclassified

Chemical Exchange Saturation Transfer (CEST) Agents: Quantum Chemistry and MRI

Feng

Zhu

et al. 2015

Chemistry A European J

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Diamagnetic chemical exchange saturation transfer (CEST) contrast agents offer an alternative to Gd3+-based contrast agents for MRI. They are characterized by containing protons that can rapidly exchange with water and it is advantageous to have these protons resonate in a spectral window that is far removed from water. Here, we report the first results of DFT calculations of the 1H nuclear magnetic shieldings in 41 CEST agents, finding that the experimental shifts can be well predicted (R2 = 0.882). We tested a subset of compounds with the best MRI properties for toxicity and for activity as uncouplers, then obtained mice kidney CEST MRI images for three of the most promising leads finding 16 (2,4-dihydroxybenzoic acid) to be one of the most promising CEST MRI contrast agents to date. Overall, the results are of interest since they show that 1H NMR shifts for CEST agents—charged species—can be well predicted, and that several leads have low toxicity and yield good in vivo MR images.

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“…Although single nucleoside species have been used as general IS for all modifications in a given sample ( 19 , 25 ), we maintain that accurate response factors can only derive from isotopomers for each specific modification ( 26 ), obtained by stable isotope labeling (SIL) ( 27 – 29 ). The Carell laboratory has recently published impressive quantification results ( 30 , 31 ) based on a series of isotope-labeled internal standards obtained by means of synthetic organic chemistry. By this approach, determination of absolute substance amounts in samples is possible because the isotopomers are available in quantities sufficient for accurate weighing.…”

Section: Introductionmentioning

confidence: 99%

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

Kellner

Ochel

Thüring

et al. 2014

Nucleic Acids Research

120

105

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In the resurging field of RNA modifications, quantification is a bottleneck blocking many exciting avenues. With currently over 150 known nucleoside alterations, detection and quantification methods must encompass multiple modifications for a comprehensive profile. LC–MS/MS approaches offer a perspective for comprehensive parallel quantification of all the various modifications found in total RNA of a given organism. By feeding 13C-glucose as sole carbon source, we have generated a stable isotope-labeled internal standard (SIL-IS) for bacterial RNA, which facilitates relative comparison of all modifications. While conventional SIL-IS approaches require the chemical synthesis of single modifications in weighable quantities, this SIL-IS consists of a nucleoside mixture covering all detectable RNA modifications of Escherichia coli, yet in small and initially unknown quantities. For absolute in addition to relative quantification, those quantities were determined by a combination of external calibration and sample spiking of the biosynthetic SIL-IS. For each nucleoside, we thus obtained a very robust relative response factor, which permits direct conversion of the MS signal to absolute amounts of substance. The application of the validated SIL-IS allowed highly precise quantification with standard deviations <2% during a 12-week period, and a linear dynamic range that was extended by two orders of magnitude.

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Total Synthesis of the Hypermodified RNA Bases Wybutosine and Hydroxywybutosine and Their Quantification Together with Other Modified RNA Bases in Plant Materials

Cited by 16 publications

References 33 publications

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA

Chemical Exchange Saturation Transfer (CEST) Agents: Quantum Chemistry and MRI

Absolute and relative quantification of RNA modifications via biosynthetic isotopomers

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