Characterization of Modified RNA by Top‐Down Mass Spectrometry

Taucher, Monika; Breuker, Kathrin

doi:10.1002/anie.201206232

Cited by 66 publications

(66 citation statements)

References 35 publications

(26 reference statements)

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“…While mass spectrometry, in general, and LC-ESI-MS/MS, in particular, are powerful analytical platforms for mapping tRNA modifications, direct analysis of intact tRNAs is typically beyond the capabilities of common mass spectrometry instrumentation requiring specialized high-end capabilities [43]. Thus, an analytical strategy was developed by the McCloskey group, which utilizes base-specific ribonucleases (RNases) to digest tRNAs into smaller oligonucleotides (or digestion products), which are more amenable to common LC-ESI-MS/MS or MALDI-MS systems [44,45].…”

Section: The Bottom-up Approach To Rna Modification Mappingmentioning

confidence: 99%

Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

2017

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Liquid chromatography, coupled with tandem mass spectrometry, has become one of the most popular methods for the analysis of post-transcriptionally modified transfer ribonucleic acids (tRNAs). Given that the information collected using this platform is entirely determined by the mass of the analyte, it has proven to be the gold standard for accurately assigning nucleobases to the sequence. For the past few decades many labs have worked to improve the analysis, contiguous to instrumentation manufacturers developing faster and more sensitive instruments. With biological discoveries relating to ribonucleic acid happening more frequently, mass spectrometry has been invaluable in helping to understand what is happening at the molecular level. Here we present a brief overview of the methods that have been developed and refined for the analysis of modified tRNAs by liquid chromatography tandem mass spectrometry.

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Section: The Bottom-up Approach To Rna Modification Mappingmentioning

confidence: 99%

Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

2017

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“…2c left). Characterization of the AIEX-purified MLV PK by top-down Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) 20,21 revealed a high sample homogeneity (Fig. 2c right), the correct sequence, and localized the SI label to A17 (ESI, † Fig.…”

Section: -13mentioning

confidence: 99%

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

et al. 2017

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We showcase the high potential of the 2 0 -cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectro- Solution and solid state nuclear magnetic resonance (NMR) spectroscopy have proven to be highly suitable to address structural and dynamic features of RNA. 1-4 A prerequisite to apply state-of-the-art NMR experiments is the introduction of a stable isotope (SI) labelling pattern using 13 C/ 15 N labelled RNA or DNA precursors. [5][6][7][8] The most wide-spread method uses labelled (2 0 -deoxy)-ribonucleotide triphosphates and enzymes to produce the desired RNA or DNA sequence enriched with 13 C and 15 N nuclei. 1,5 This approach enables to produce sufficient amounts of RNA and DNA for NMR spectroscopic applications. This well-established method allows nucleotide specific labeling by mixing a SI-labeled with unlabeled d/rNTPs. Especially in larger RNAs (460 nt) such nucleotide specific SI-labeling can still lead to significant resonance overlap. That is why, the PLOR (position-selective labelling of RNA) method was recently introduced, which holds the promise to site-specifically label RNA using SI-labelled ribonucleotide triphosphates and T7 RNA polymerase. 9 An alternative method was concurrently developed making use of the synthesis of 2 0 -O-tri-iso-propylsilyloxymethylphosphoramidites and solid phase synthesis. 10-13The approach works well for medium sized RNAs up to 50 nts and the synthetic access to the SI-labelled building blocks is well established.10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts. Due to this restriction, large RNAs are only accessible via enzymatic ligation strategies using T4 RNA/DNA ligase making extra optimization steps necessary or introducing new problems, such as finding the optimal ligation site or issues regarding up-scaling and yield of the ligation product. 14-16 Thus, an improved synthetic procedure to directly address SI-labelling of larger RNAs (460 nt) at amounts suitable for NMR would be highly desirable. We report the synthesis of SI-labelled RNAs ranging in size between 60 to 80 nts capitalizing on the 2 0 -cyanoethoxymethyl (CEM) RNA synthesis method. 17,18 As these CEM building blocks are not commercially available all phosphoramidites were produced in-house and we further synthesized 13 C-/ 15 N-labelled unmodified and naturally occurring modified RNA phosphoramidites (Fig. 1a and b). In detail, we focused on the synthesis of 8- We used these monomer units to produce SI-labelled RNAs exceeding the size limitation of 60 nucleotides for NMR up to 20 nucleotides. The RNAs reported here were synthesized on a 1.3 mmol scale and on a 1000...

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“…Because top-down MS does not involve hydrolysis, it can be used to detect and characterize different forms of a protein or RNA whereby it can provide a complete description of the primary structure and reveal all mass-altering modifications, as well as any correlations that exist between these modifications (43). Top-down MS (43–45) has been used for the direct identification of human cellular microRNA (miRNA) (46), small interfering RNA (siRNA) (47), for the characterization of synthetic 21–23 nt RNA (48), tRNA (49) and RNA stem–loop motifs modified by structural probes (50), for de novo sequencing of tRNA (51), and for probing tat peptide binding to TAR RNA (52). …”

Section: Introductionmentioning

confidence: 99%

“…Here we explore the potential of top-down MS for the simultaneous identification, localization and relative quantitation of methylated RNA residues using electrospray ionization (ESI), low-energy collisionally activated dissociation (CAD), and electron detachment dissociation (EDD) (51,53,54). Our label-free, direct approach for this purpose builds on previous studies of modified bovine ribonuclease A (55), human histone H3 (56,57) and H4 (58), tropomyosin (59), and cardiac troponin I (60) proteins, and requires that RNA methylation does not affect RNA desorption, ionization and dissociation.…”

Section: Introductionmentioning

confidence: 99%

Label-free, direct localization and relative quantitation of the RNA nucleobase methylations m6A, m5C, m3U, and m5U by top-down mass spectrometry

Glasner

Riml

Micura

et al. 2017

Nucleic Acids Research

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Nucleobase methylations are ubiquitous posttranscriptional modifications of ribonucleic acids (RNA) that can substantially increase the structural diversity of RNA in a highly dynamic fashion with implications for gene expression and human disease. However, high throughput, deep sequencing does not generally provide information on posttranscriptional modifications (PTMs). A promising alternative approach for the characterization of PTMs, i.e. their identification, localization, and relative quantitation, is top-down mass spectrometry (MS). In this study, we have investigated how specific nucleobase methylations affect RNA ionization in electrospray ionization (ESI), and backbone cleavage in collisionally activated dissociation (CAD) and electron detachment dissociation (EDD). For this purpose, we have developed two new approaches for the characterization of RNA methylations in mixtures of either isomers of RNA or nonisomeric RNA forms. Fragment ions from dissociation experiments were analyzed to identify the modification type, to localize the modification sites, and to reveal the site-specific, relative extent of modification for each site.

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Characterization of Modified RNA by Top‐Down Mass Spectrometry

Cited by 66 publications

References 35 publications

Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

Mapping Post‐Transcriptional Modifications onto Transfer Ribonucleic Acid Sequences by Liquid Chromatography Tandem Mass Spectrometry

Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

Label-free, direct localization and relative quantitation of the RNA nucleobase methylations m6A, m5C, m3U, and m5U by top-down mass spectrometry

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