Identification of a T7 RNA polymerase variant that permits the enzymatic synthesis of fully 2'-O-methyl-modified RNA.

•A single amino acid substitution converts wildtype T7 RNA polymerase into a processive 2′-O-methyl nucleotide polymerase.•Residue R425 is located at the end of a sequence motif (DX2GR) which is conserved among many DNA and RNA polymerases.•An all-2′-O-methyl-modified aptamer analog retains function by binding the cognate ligand of its natural antetype.