A solid-phase enzymatic synthesis platform for the facile production of 2′-fluoroarabinonucleic acid (FANA) and chimeric XNA oligonucleotides using an evolved XNA polymerase

Xenobiotic nucleic acids (XNAs) significantly expand the range of genetic polymers and serve as promising alternatives to DNA and RNA for numerous biological applications. However, the extensive exploration and application of XNAs are limited by low sustainability and yields in solid-phase oligonucleotide synthesis, as well as by the unavailability of efficient XNA polymerases for polymerase-mediated XNA production. To address the limitations in XNA production, we developed a solid-phase enzymatic XNA oligonucleotide synthesis platform using a laboratory-evolved XNA polymerase, SFM5-7, which exhibits excellent activity for synthesizing DNA, RNA, 2'-fluoroarabinonucleic acid (FANA), and other 2'-modified XNA oligonucleotides. This platform employs ribonucleotide insertion and alkaline cleavage of the oligonucleotide product before and after SFM5-7-mediated XNA synthesis, enabling recycled XNA synthesis through the reuse of a bead-immobilized self-priming hairpin DNA template. The platform's potential and versatility are demonstrated by the production of FANA, 2'-modified RNAs, chimeric XNAs, 5'-end-labeled FANA, and an active FANAzyme. This platform should facilitate the customized production of functional XNAs with programmable modifications, accelerating their applications in biotechnology and biomedicine.