Radical Transfer Dissociation for De Novo Characterization of Modified Ribonucleic Acids by Mass Spectrometry

Calderisi, Giovanni; Glasner, Heidelinde; Breuker, Kathrin

doi:10.1002/ange.201914275

Cited by 6 publications

(7 citation statements)

References 24 publications

(27 reference statements)

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“…The highest resolution sequencing method, such as TAIL-seq (40), PAlso-seq (41,42), and FLAMseq (43) provide information on nucleotide sequence, polyA tail length, and the presence of adenine modifications. Mass spectrometry has been successfully used in the analysis of RNA, including mRNA, either by direct infusion analysis or combined with chromatographic techniques (44)(45)(46)(47)(48)(49)(50)(51). This powerful technique provides information on RNA structure and composition, including polyA tail length.…”

Section: Introductionmentioning

confidence: 99%

Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression

Strzelecka

Śmietański

Sikorski

et al. 2020

RNA

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Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases-SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'mononucleotides. The method also estimated the average polyA tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the polyA tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.

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Section: Introductionmentioning

confidence: 99%

Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression

Strzelecka

Śmietański

Sikorski

et al. 2020

RNA

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“…Sequence coverage describes the number of cleavage sites where at least one resulting product could be detected. There are a fair number of techniques used for this method (CAD [ 98 ], EDD [ 99 ], RTD [ 100 ], AINETD [ 101 ], etc.) but the overall methodology remains the same: Fragment the structured ion and analyze the fragments to achieve overlapping coverage for the sequence.…”

Section: Experiments That Can Help Validate MD Simulation Resultsmentioning

confidence: 99%

Challenges with Simulating Modified RNA: Insights into Role and Reciprocity of Experimental and Computational Approaches

D'Esposito

Myers

Chen

et al. 2022

Genes

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RNA is critical to a broad spectrum of biological and viral processes. This functional diversity is a result of their dynamic nature; the variety of three-dimensional structures that they can fold into; and a host of post-transcriptional chemical modifications. While there are many experimental techniques to study the structural dynamics of biomolecules, molecular dynamics simulations (MDS) play a significant role in complementing experimental data and providing mechanistic insights. The accuracy of the results obtained from MDS is determined by the underlying physical models i.e., the force-fields, that steer the simulations. Though RNA force-fields have received a lot of attention in the last decade, they still lag compared to their protein counterparts. The chemical diversity imparted by the RNA modifications adds another layer of complexity to an already challenging problem. Insight into the effect of RNA modifications upon RNA folding and dynamics is lacking due to the insufficiency or absence of relevant experimental data. This review provides an overview of the state of MDS of modified RNA, focusing on the challenges in parameterization of RNA modifications as well as insights into relevant reference experiments necessary for their calibration.

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“…Recently, a new dissociation technique named radical transfer dissociation (RTD) was reported [72]. For this method, cobalt(III)hexamine ([Co III (NH 3 ) 6 ] 3+ ) was utilized as a solution additive to form complexes with RNA.…”

Section: Detection Of Nucleic Acidsmentioning

confidence: 99%

“…Reprinted (adapted) with permission from Calderisi et al. [72]. Copyright (2020) John Wiley and Sons…”

Section: Detection Of Nucleic Acidsmentioning

confidence: 99%

Recent developments in the characterization of nucleic acids by liquid chromatography, capillary electrophoresis, ion mobility, and mass spectrometry (2010–2020)

Santos

Brodbelt

2020

J of Separation Science

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The development of new strategies for the analysis of nucleic acids has gained momentum due to the increased interest in using these biomolecules as drugs or drug targets. The application of new mass spectrometry ion activation techniques and the optimization of separation methods including liquid chromatography, capillary electrophoresis, and ion mobility have allowed more detailed characterization of nucleic acids and oligonucleotide therapeutics including confirmation of sequence, localization of modifications and interaction sites, and structural analysis as well as identification of failed sequences and degradation products. This review will cover tandem mass spectrometry methods as well as the recent developments in liquid chromatography, capillary electrophoresis, and ion mobility coupled to mass spectrometry for the analysis of nucleic acids and oligonucleotides.

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Radical Transfer Dissociation for De Novo Characterization of Modified Ribonucleic Acids by Mass Spectrometry

Cited by 6 publications

References 24 publications

Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression

Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression

Challenges with Simulating Modified RNA: Insights into Role and Reciprocity of Experimental and Computational Approaches

Recent developments in the characterization of nucleic acids by liquid chromatography, capillary electrophoresis, ion mobility, and mass spectrometry (2010–2020)

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