Advancing the enzymatic toolkit for 2'-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.

2'-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5'-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3' to 5' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling