However, these applications require the modification of RNA which otherwise shows poor resistance to cellular nucleases. To this end, typically the ribose 2´-hydroxyl moieties are substituted by O-methyl groups. This step is hampered by the fact that wild-type RNA-Polymerase is inefficient in incorporating modified nucleotides. Although mutant enzymes have been engineered which are able to insert some or even all 2´-O-me-modified nucleotides, none of the enzymes present so far is able to synthesize transcripts of sufficient lengths. By an in-depth analysis of the T7-RNA-Polymerase and protein engineering, the inventors were able to create a variant which is both a generalist polymerase capable of incorporating all 2´-O-me-modified nucleotides as well as synthesizing fully modified RNA of up to approx. 1,000 nucleotides.
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