Herein we describe results on the pairing properties of synthetic DNA and RNA oligonucleotides that contain nucleotide analogues with a 7-membered sugar ring (oxepane nucleotides). Specifically, we describe the stereoselective synthesis of a set of three oxepane thymine nucleosides (OxT), their conversion to phosphoramidite derivatives, and their use in solid-phase synthesis to yield chimeric OxT-DNA and OxT-RNA strands. The different regioisomeric OxT phosphoramidites allowed for positional variations of the phosphate bridge and assessment of duplex stability when the oxepane nucleotides were incorporated in dsDNA, dsRNA, and DNA-RNA hybrids. Little to no destabilization was observed when two of the three regioisomeric OxT units were incorporated in the DNA strand of DNA-RNA hybrids, a remarkable result considering the dramatically different structure of oxepanes in comparison to 2'-deoxynucleosides. Extensive molecular modeling and dynamics studies further revealed the various structural features responsible for the tolerance of both OxT modifications in DNA-RNA duplexes, such as base-base stacking and sugar-phosphate H-bond interactions. These studies suggest that oxepane nucleotide analogues may find applications in synthetic biology, where synthetic oligonucleotides can be used to create new tools for biotechnology and medicine.