Impact of Single Acyclic Phosphonate Nucleotide (ZNA) Modifications on DNA Duplex Stability

An acyclic phosphonate‐linked nucleic acid backbone (ZNA) demonstrated the capability to support duplex formation and propagate genetic information in vivo, unveiling its potential for evolution into a synthetic genetic system (XNA). To determine the structural impact of such modification, modified Dickerson Drew DNA dodecamers (DDDs) were prepared by solid phase synthesis, each containing either an (R) or (S) isomeric form of a cytosine ZNA nucleotide. While the DDD is known to adopt a stable duplex, both duplex and hairpin forms were simultaneously observed for both modified oligonucleotides by NMR spectroscopy over a broad temperature range (5–65 °C). Diffusion‐ordered spectroscopy (DOSY) experiments allowed to separate duplex and hairpin signals based on the different diffusion constants of both conformational states. For the oligomer containing (R)‐ZNA, only the duplex form occurred at 5 °C, while it was not possible to determine by NMR a single hairpin conformation at higher temperatures. In the case of the (S)‐ZNA nucleoside modified oligomer, both hairpin and duplex forms were observable at 0 °C, while a single hairpin conformation was detected at 37 °C, suggesting a higher destabilizing effect on dsDNA.