TY  - BOOK
AU  - Freund,Niklas
AU  - Taylor,Alexander I.
AU  - Arangundy-Franklin,Sebastian
AU  - Subramanian,Nithya
AU  - Peak-Che
AU  - Whitaker,Amy M.
AU  - Freudenthal,Bret D.
AU  - Abramov,Mikhail
AU  - Herdewijn,Piet
AU  - Holliger,Philipp
TI  - A two-residue nascent strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA
PY  - 2022///-10-13
KW  - Text
KW  - local
N1  - /pmc/articles/PMC7614059; /pubmed/36229679
N2  - Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2'-modified nucleic acids, which are of interest in nucleic acid-based drug development. Here we describe the discovery of a two-residue, nascent strand, steric control "gate" in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously-described RNA polymerase activity unlocks the synthesis of 2'-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750 nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2'OMe-RNA ("2'OMezymes") for the allele-specific cleavage of oncogenic KRAS (G12D) and β-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2'OMe- / MOE-RNA aptamers with high affinity for Vascular Endothelial Growth Factor. Our results open up these 2'-modified RNAs-used in several approved nucleic acid therapeutics-for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology
UR  - http://dx.doi.org/10.1038/s41557-022-01050-8
ER  -