Efficient Chemical Synthesis and Enzymatic Replication of 3′-Deoxyapionucleic Acid (ApioNA): An RNA-Selective XNA with Conformational Flexibility

Xeno-nucleic acids (XNAs) have emerged as versatile platforms due to their sugar scaffolds that enable precise chemical tuning, leading to distinct classes ranging from fully compatible systems to orthogonal systems. However, sugar-modified XNAs capable of discriminating between RNA and DNA have remained elusive. In this work, we establish 3′-deoxyapionucleic acid (ApioNA) as a structurally flexible XNA with inherent RNA-selective hybridization properties. We achieved efficient production of ApioNA oligonucleotides through an efficient asymmetric synthesis of their building blocks and systematically characterized their properties. Thermal melting analyses demonstrate that ApioNA forms moderately stable duplexes with RNA while exhibiting weak pairing with DNA and negligible self-association. Quantum mechanical calculations reveal that the apiofuranose ring populates two low-energy puckering states, and the addition of an extra methylene group enhances backbone flexibility, thereby favoring A-form geometry for RNA pairing while requiring a higher-energy state to accommodate B-form DNA. Despite its weak affinity for DNA, ApioNA supports a complete enzymatic replication cycle using commercial Therminator and Bst 3.0 polymerases with approximately 98% fidelity and is processed by native E. coli machinery in vivo. This work fills the RNA-selective niche in the XNA functional landscape and establishes sugar puckering dynamics and backbone flexibility as key design principles for programming nucleic acid pairing specificity.