Engineered polymerase-mediated efficient synthesis of site-specifically functionalized RNA and 2'-modified RNA oligonucleotides via genetic alphabet expansion.

2'-Modified RNA oligonucleotides have found broad use in biotechnology and biomedicine, requiring their efficient synthesis and controllable labelling. While engineered polymerases enable their synthesis, controllable labelling methods remain underdeveloped. Unnatural base pairs (UBPs) have been developed to expand the genetic alphabet, leading to not only the creation of semi-synthetic organisms, but also technologies for site-specific labelling of DNA and RNA. Herein we demonstrate that SFM4-6, an engineered polymerase optimized for 2'-modified RNA oligonucleotide synthesis, can efficiently synthesize unnatural base pair dNaM (2-methoxy-3-(2'-deoxy-β-D-erythro-pentofuranosyl)-naphthalene)-dTPT3 ((2'-deoxy-β-D-erythro-pentofuranosyl)-thieno[3,4]pyridine-2-thione) and its analogues, enabling site-specific incorporation of functionalized unnatural base-containing nucleotides into RNA and 2'-modified RNA oligonucleotides. By combining this with a strategy involving controlled pause and restart of primer extension, we establish methods for producing single or dual-labelled RNA and 2'-modified RNA oligonucleotides. These methods successfully produce various functional labelled RNA and 2'-modified RNA oligonucleotides, highlighting their broad applicability in nucleic acid research and biotechnology.