Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of alpha-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.