# 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

## 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.

## Key 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 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.