No backbone motif other than phospho-ribose and phospho-deoxyribose has been found in natural nucleic acids, currently restricting the molecular types of replicable biopolymers to DNA and RNA. With the aim of propagating and expressing a third type of nucleic acid in vivo, we assessed the replicability of polynucleotides with a phospho-hexitol backbone (HNA) in vivo and in vitro. Faithful polymerisation of up to four deoxynucleotides templated by hexitol oligonucleotides was established in vitro using DNA polymerase from Escherichia coli (PolA Klenow exo-fragment) and Thermus aquaticus (Taq polymerase). Condensation of up to three successive hTTPs (hexitol thymidine triphosphate) in responses to a pentameric hexitol template (hA)5 could also be demonstrated in vitro. Such a marginal HNA-dependent HNA polymerase activity of natural polymerases may be evolved in the future to catalyse in vitro amplification of HNA. The transmission of a two-codon-long genetic message carried on a hexameric hexitol template was also established using a selection screen for restoring thymidylate synthase activity in E. coli. These results exemplify the potential that can be explored by converting artificial substrates with natural enzymes in the field of informational polymer synthesis.