Enzymatic Synthesis of Modified RNA Containing 5‑Methyl- or 5‑Ethylpyrimidines or Substituted 7‑Deazapurines and Influence of the Modifications on Stability, Translation, and CRISPR-Cas9 Cleavage
Tania Sanchez-Quirante, Erika Kužmová, Miguel Riopedre-Fernandez, Sebastian Golojuch, Pavel Vopálenský, Veronika Raindlová, Afaf H. El-Sagheer, Tom Brown, Michal Hocek
TL;DR
Researchers synthesized modified RNA with specific base changes and tested how these modifications affect RNA stability, translation, and CRISPR-Cas9 gene editing.
Contribution
The study introduces new enzymatic methods to synthesize modified RNA and evaluates their impact on translation and CRISPR-Cas9 cleavage.
Findings
Modified RNA with 5-methyluracil and -cytosine enhanced in vitro translation, while 7-deazaadenines inhibited it.
In cellulo translation was significantly enhanced by 7-deazaguanine and moderately by 5-methyl- or 5-ethylcytosine.
7-alkyl-7-deazaadenines completely inhibited CRISPR-Cas9 gene cleavage.
Abstract
A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement…
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Taxonomy
TopicsBiochemical and Molecular Research · CRISPR and Genetic Engineering · HIV/AIDS drug development and treatment