000 02203 am a22003013u 4500
042 _adc
100 1 0 _aFreund, Niklas
_eauthor
_92248
700 1 0 _aTaylor, Alexander I.
_eauthor
_91456
700 1 0 _aArangundy-Franklin, Sebastian
_eauthor
_92249
700 1 0 _aSubramanian, Nithya
_eauthor
_92250
700 1 0 _aPeak-Che
_eauthor
_91459
700 1 0 _aWhitaker, Amy M.
_eauthor
_92251
700 1 0 _aFreudenthal, Bret D.
_eauthor
_92252
700 1 0 _aAbramov, Mikhail
_eauthor
_92253
700 1 0 _aHerdewijn, Piet
_eauthor
_92254
700 1 0 _aHolliger, Philipp
_eauthor
_91460
245 0 0 _aA two-residue nascent strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA
260 _c2022-10-13.
500 _a/pmc/articles/PMC7614059/
500 _a/pubmed/36229679
520 _aSteric 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.
540 _a
546 _aen
690 _aArticle
655 7 _aText
_2local
786 0 _nNat Chem
856 4 1 _uhttp://dx.doi.org/10.1038/s41557-022-01050-8
_zConnect to this object online.
999 _c2212
_d2212