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1. 5-ethynyl uridine labeling of nascent mitochondrial genome transcription v1

   https://doi.org/10.17504/protocols.io.261gero6wl47/v1  

   Waingankar, Tejashree Pradip; Lewis, Samantha C (November 2024, Springer Nature)

   This protocol details a method for 5-ethynyl uridine labeling of nascent mitochondrial genome transcription coupled with immunofluorescence detection of mtDNA-binding proteins in cultured cells. The result is a fluorescent readout of localized mitochondrial gene transcription compatible with high-resolution microscopy. 

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2. Synthesis of atom-specific nucleobase and ribose labeled uridine phosphoramidite for NMR analysis of large RNAs

   https://doi.org/10.1007/s00706-021-02851-2  

   Olenginski, Lukasz T.; Becette, Owen B.; Beaucage, Serge L.; Dayie, Theodore K. (November 2021, Monatshefte für Chemie - Chemical Monthly)
3. Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

   https://doi.org/10.1039/D2CB00229A  

   Jones, Joshua D.; Franco, Monika K.; Smith, Tyler J.; Snyder, Laura R.; Anders, Anna G.; Ruotolo, Brandon T.; Kennedy, Robert T.; Koutmou, Kristin S. (May 2023, RSC Chemical Biology)

   Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N 2-methylguanosine, N 2, N 2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N 2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context. 

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4. Cyano Modification on Uridine Decreases Base-Pairing Stability and Specificity through Neighboring Disruption in RNA Duplex

   https://doi.org/10.1002/cbic.201800399  

   Mao, Song; Ranganathan, Srivathsan V.; Tsai, Hsu-Chun; Haruehanroengra, Phensinee; Shen, Fusheng; Valsangkar, Vibhav A.; Han, Bo; Hassan, Abdalla E.; Chen, Alan; Sheng, Jia (December 2018, ChemBioChem)
5. Increased Photostability of the Integral mRNA Vaccine Component N ₁ ‐Methylpseudouridine Compared to Uridine

   https://doi.org/10.1002/chem.202103667  

   Hoehn, Sean_J; Krul, Sarah_E; Skory, Brandon_J; Crespo‐Hernández, Carlos_E (December 2021, Chemistry – A European Journal)

   Abstract N₁‐Methylation of pseudouridine (m¹ψ) replaces uridine (Urd) in several therapeutics, including the Moderna and BioNTech‐Pfizer COVID‐19 vaccines. Importantly, however, it is currently unknown if exposure to electromagnetic radiation can affect the chemical integrity and intrinsic stability of m¹ψ. In this study, the photochemistry of m¹ψ is compared to that of uridine by using photoirradiation at 267 nm, steady‐state spectroscopy, and quantum‐chemical calculations. Furthermore, femtosecond transient absorption measurements are collected to delineate the electronic relaxation mechanisms for both nucleosides under physiologically relevant conditions. It is shown that m¹ψ exhibits a 12‐fold longer¹ππ* decay lifetime than uridine and a 5‐fold higher fluorescence yield. Notably, however, the experimental results also demonstrate that most of the excited state population in both molecules decays back to the ground state in an ultrafast time scale and that m¹ψ is 6.7‐fold more photostable than Urd following irradiation at 267 nm. 

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