双残基新生链空间门控制 2′-O-甲基-和 2′-O-(2-甲氧基乙基)-RNA 的合成
Nature Chemistry
(
IF19.2
)
Pub Date : 2022-10-13, DOI: 10.1038/s41557-022-01050-8
Niklas Freund
1
,
Alexander I Taylor
1,
2
,
Sebastian Arangundy-Franklin
1
,
Nithya Subramanian
1
,
Sew-Yeu Peak-Chew
1
,
Amy M Whitaker
3,
4
,
Bret D Freudenthal
3
,
Mikhail Abramov
5
,
Piet Herdewijn
5
,
Philipp Holliger
1
Affiliation
MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK.
Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge, UK.
Laboratory of Genome Maintenance and Structural Biology, Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA.
Cancer Epigenetics Institute, Fox Chase Cancer Center, Philadelphia, PA, USA.
Medicinal Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium.
A two-residue nascent-strand steric gate controls synthesis of 2′-O-methyl- and 2′-O-(2-methoxyethyl)-RNA
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.