In Vitro transcriptase: Methods for studying XNA synthesis

Xeno nucleic acids (XNAs), also known as unnatural nucleic acids, are analogs of RNA and DNA featuring chemical modifications such as altered sugar backbones. These modifications confer enhanced nuclease resistance, novel or stabilized structural motifs, and potentially improved binding affinity to biomolecule targets. Therefore, XNAs are versatile candidates for incorporation into oligonucleotide therapeutics such as ASOs and siRNAs. XNA can be synthesized by solid-phase oligonucleotide synthesis or by in vitro transcription using engineered polymerases. In biology labs without a solid-phase oligo synthesizer, in vitro transcription of XNA is an effective method to synthesize and utilize XNAs. Here we describe the purification of an engineered polymerase SFM 5-7 and an in vitro transcription protocol of XNA using this enzyme. SFM 5-7 has been reported to synthesize RNA analogues with 2'-modifications, including 2'-O-Me and 2'-F. In this protocol, we use 2'-F-RNA as an example. Denaturing Polyacrylamide Gel Electrophoresis (PAGE) is used to monitor the reaction.