Previously, a series of three poly(amidoamine)s was designed and synthesized by polymerizing oxylate, succinate, or adipate groups With These resulting polymers (named O4, S4, and A4, respectively) were created as models to poly(glycoamidoamine) nucleic acid delivery agents to understand how the absence of hydroxyl groups and changes in the amide bond spacing affect polymer degradation, plasmid DNA (pDNA) encapsulation, toxicity, and transfection efficiency in vitro To understand differences in the biological properties quantitatively, we investigated the mechanism of interaction between these macromolecules and pDNA to reveal differences in pDNA binding affinity and complexation as I function of structure Herein, several analytical techniques such as dynamic light scattering, circular dichroism, thermal gravimetric analysis, isothermal nitration calorimetry (ITC), and ethidium bromide exclusion assays were used to examine the pDNA binding strength of O4, S4, and A4, and the results are compared with the previous series of poly(glycoamidoamine)s. It was found that the length of the amide bond spacer in these nonhydroxylated analogs did affect the pDNA binding affinity to a small degree (binding affinity order A4 > S4 > O4). The increase in binding affinity with longer methylene spacer was riot due to hydrophobic interactions but likely from optimization in electrostatic interactions and hydrogen bond formation Even though O4 was revealed to have the lowest pDNA binding affinity of the nonhydroxylated series. this polymer yields the highest cellular transfection efficiency, which is likely an effect of the faster hydrolysis rate.