2025/11/06 by Tania Sanchez-Quirante, Erika Kužmová, Miguel Riopedre-Fernández +6 · 1 voice
Biochemistry, Genetics and Molecular Biology · Medicine · #Biochemical and Molecular Research #CRISPR and Genetic Engineering #HIV/AIDS drug development and treatment
paper · pdf · doi:10.1021/acschembio.5c00692
openalex created_date 2025/11/06 · openalex publication_date 2025/11/06 · openalex updated_date 2026/07/02
High Resolution Image Download MS PowerPoint Slide 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 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.