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

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 <i>in vitro</i> transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding <i>Renilla</i> luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in <i>in vitro</i> and <i>in cellulo</i> translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the <i>in vitro</i> translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, <i>in cellulo</i> experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.