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. Sterically demanding 2′-modified nucleotides used in antisense therapeutics have thus far been challenging to synthesise enzymatically. Now, it has been shown that mutation of two gatekeeper residues in an archaeal DNA polymerase unlocks efficient synthesis of the modified nucleic acid oligomers 2′-O-methyl-RNA and 2′-O-(2-methoxyethyl)-RNA and enables the evolution of 2′-O-methyl-RNA enzymes.