The structural and thermodynamic origins of the destabilization of a backbone-modified DNA duplex 1, formed between d(CpGpT(N)TpGpC), containing a single aminoethyl group (-CH2-CH2-NH2+-) in place of the phosphodiester (-O-PO2--O-) linkage of the central TT dimer, and d(GpCpApApCpG) are investigated. Analyses for the corresponding native duplex and two other related structural analogues of duplex 1 have been compared. Duplex 1 shows a cooperative thermal melting transition that is consistent with a two-state process. At a 2 mM concentration, the melting temperature of duplex 1 is reduced by 17 degrees C from the native duplex, and this decrease in stability is further assigned to an unfavorable decrease in enthalpy of 7 kcal mol(-1) and a favorable increase in entropy of 15 eu mol(-1) NMR structural analysis shows that the modified duplex 1 still adopts a canonical B-DNA conformation with Watson-Crick base pairing preserved; however, the CH2 group that replaces the native PO2- group in the modified backbone is flexible and free to collapse onto a hydrophobic core formed by the base edges and sugar rings of the flanking TT/AA nucleosides of the duplex. This conformation is significantly different from the maximally solvent-exposed orientation of the native phosphate in DNA. The entropic origin of the 15 eu mol(-1) difference between the native and the modified duplex 1 is attributed to the hydrophobic interaction between the collapsed ethylamine Linkage with the hydrophobic core of duplex 1. This assignment is further supported by a favorable comparison between the observed change in entropy and the estimated value for the hydrophobic interaction around the modified region. This estimated value is based on recent experimental measurement of the hydrophobic interaction between aliphatic groups and nucleic acids as well as ethylamine solvent transfer data. The overall decrease in the stability of duplex 1 results from a decrease in base stacking and hydrogen-bonding interactions between the base pairs. A model is, therefore, proposed to explain how the change in the local backbone conformation could disrupt the long-range cooperativity of DNA duplex formation upon backbone modifications. These studies provide an approach for identifying the factors that control the stability of the nucleic acid duplex structures containing backbone modifications, with direct implication for designing antisense oligonucleotides and template-directed reactions containing non-native phosphodiester linkages.