The physical properties of a DNA:RNA hybrid sequence d(CCAACGTTGG)·(CCAACGUUGG) with modifications at the C2'-positions of the DNA strand by 2'-O-methyl (OMe) and 2'-S-methyl (SMe) groups are studied using computational techniques. Molecular dynamics simulations of SMe-DNA:RNA, OMe-DNA:RNA and standard DNA:RNA hybrids in explicit water indicate that the nature of the C2'-substituent has a significant influence on the macromolecular conformation. While the RNA strand in all duplexes maintains a strong preference for C3'-endo sugar puckering, the DNA strand shows considerable variation in this parameter depending on the nature of the C2'-substituent. In general, the preference for C3'-endo puckering follows the following trend: OMe-DNA>DNA>SMe-DNA. These results are further corroborated using ab initio methods. Both gas phase and implicit solvation calculations show the C2'-OMe group stabilizes the C3'-endo conformation while the less electronegative SMe group stabilizes the C2'-endo conformation when compared to the standard nucleoside. The macromolecular conformation of these nucleic acids also follows an analogous trend with the degree of A-form character decreasing as OMe-DNA:RNA>DNA:RNA>SMe-DNA:RNA. A structural analysis of these complexes is performed and compared with experimental melting point temperatures to explain the structural basis to improved binding affinity across this series. Finally, a possible correlation between RNase H activity and conformational changes within the minor groove of these complexes is hypothesized.