Epitranscriptomics: The new RNA code and the race to drug it

Cross, Ryan

doi:10.1021/cen-09707-cover

Cited by 5 publications

(5 citation statements)

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“…Support for this is the use of Ψ or its N1-methyl derivative for this purpose in mRNA vaccines 56 as well as the possibility that Ψ serves the same function in HIV and flaviviruses. 57 , 58 If Ψ is used by SARS-CoV-2 and possibly other coronaviruses during the infection cycle to minimize immune stimulation, this points to new schemes for intervention; 59 additionally, if Ψ is found to be essential for infectivity of the virus, an inspection of host genetic variants for pseudouridine synthase enzymes that impact activity may reveal more clues as to why host outcomes from SARS-CoV-2 infection are variable beyond other comorbidity factors.…”

Section: Discussionmentioning

confidence: 99%

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2

et al. 2021

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Direct RNA sequencing for the epitranscriptomic modification pseudouridine (Ψ), an isomer of uridine (U), was conducted with a protein nanopore sensor using a helicase brake to slowly feed the RNA into the sensor. Synthetic RNAs with 100% Ψ or U in 20 different known human sequence contexts identified differences during sequencing in the base-calling, ionic current, and dwell time in the nanopore sensor; however, the signals were found to have a dependency on the context that would result in biases when sequencing unknown samples. A solution to the challenge was the identification that the passage of Ψ through the helicase brake produced a long-range dwell time impact with less context bias that was used for modification identification. The data analysis approach was employed to analyze publicly available direct RNA sequencing data for SARS-CoV-2 RNA taken from cell culture to locate five conserved Ψ sites in the genome. Two sites were found to be substrates for pseudouridine synthase 1 and 7 in an in vitro assay, providing validation of the analysis. Utilization of the helicase as an additional sensor in direct RNA nanopore sequencing provides greater confidence in calling RNA modifications.

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Section: Discussionmentioning

confidence: 99%

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2

et al. 2021

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“…[3][4][5][6][7] This is primarily due to the high sensitivity, throughput, and mass-accuracy afforded by MS. 3 In recent years, characterization of RNA oligonucleotides (oligos) by MS/MS has been incrementally adopted during the process to develop oligonucleotide therapeutics. [8][9][10][11] Furthermore, the emerging field of epitranscriptomics, which focuses on the chemical modifications of RNA molecules at a global scale, has also prompted methodological developments in MS technologies. [12][13][14][15][16][17] MS is regarded as one of the most reliable techniques to enable exploring PTMs on RNA molecules in a more efficient manner, allowing simultaneous detection of all PTMs on any modified nucleoside.…”

Section: Introductionmentioning

confidence: 99%

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

Sun

Zuo

Zhang

et al. 2023

Preprint

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Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and in epitranscriptomic studies. However, MS fragmentation behaviors of RNA oligos are understood insufficiently. In this study, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos of four to eight nucleotides (nt) in length by collision-induced dissociation (CID) using a high-resolution, accurate-mass instrument. We find that in the CID spectra acquired under the normalized collisional energy of 35%, ~70% of the total peak intensity belonged to sequencing ions (a-B, a, b, c, d, w, x, y, z), ~25% belonged to precursor ions with either complete or partial loss of a nucleobase in the form of a neutral or an anion, and the remainder were internal ions and anionic nucleobases. Of the sequencing ions, the most abundant species were y, c, w, a-B, and a ions. The charge state of the RNA precursor ions strongly affected their fragmentation behaviors. As the precursor charge increased from -1 to -5, the fractional intensity of sequencing ions in the CID spectra decreased, whereas the fractional intensity of precursor ions with neutral and/or charged losses of a nucleobase increased. Moreover, RNA oligos containing U, especially at the 3′ terminus, tended to produce precursors that lost HNCO and/or NCO-, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings build a strong foundation for mechanistic understanding of RNA fragmentation by MS/MS, contributing to future automated identification of RNA oligos from their CID spectra in a more efficient way.

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“…Mass spectrometry (MS) is a tool for elucidating the structures of biomolecules with widespread applications in biomedical research. , Using tandem mass spectrometry (MS/MS) to sequence RNAs or identify post-transcriptional modifications (PTxMs) has been attempted, − and it has seen a pressing need in recent years due to a flourish of research and development in oligoribonucleotide (oligos) therapeutic, − as well as in epitranscriptomics, which examines the chemical modifications of RNA molecules on a global scale. − MS analysis could, in theory, enable simultaneous detection of all PTxMs on any oligos, and thus, it is considered one of the most promising techniques for exploring PTxMs on RNAs …”

Section: Introductionmentioning

confidence: 99%

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

Sun

Zuo

Zhang

et al. 2023

J. Am. Soc. Mass Spectrom.

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Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a highresolution, accurate-mass instrument. We found that in CID spectra acquired under the normalized collision energy (NCE) of 35%, approximately 70% of the total peak intensity was attributed to sequencing ions (a-B, a, b, c, d, w, x, y, z), around 25% of the peak intensity came from precursor ions that experienced complete or partial loss of a nucleobase in the form of either a neutral or an anion, and the remainder were internal ions and anionic nucleobases. The top five sequencing ions were the y, c, w, a-B, and a ions. Furthermore, we observed that CID fragmentation behaviors of RNA oligos were significantly impacted by their precursor charge. Specifically, when the precursors had a charge from 1 − to 5 − , the fractional intensity of sequencing ions decreased, while that of precursors that underwent either neutral or charged losses of a nucleobase increased. Additionally, we found that RNA oligos containing 3′-U tended to produce precursors with HNCO and/or NCO − losses, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings provide valuable insights for better comprehending the mechanism behind RNA fragmentation by MS/MS, thereby facilitating the future automated identification of RNA oligos based on their CID spectra in a more efficient manner.

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Epitranscriptomics: The new RNA code and the race to drug it

Cited by 5 publications

References 0 publications

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2

Nanopore Dwell Time Analysis Permits Sequencing and Conformational Assignment of Pseudouridine in SARS-CoV-2

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

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