Establishing precise structure-activity relationships is important for the optimization of synthetic carriers for gene delivery. Sequence-defined oligomers with branched or linear shapes were synthesized to investigate the influence of topology on their biophysical properties and biological performance. Comb-like structures were synthesized consisting of an oligolysine peptide backbone modified at the ε-amino groups with four different artificial oligoamino acids, succinyl-diethylene triamine (Sdt), succinyl-triethylene tetramine (Stt), succinyl-tetraethylene pentamine (Stp), and succinyl-pentaethylene hexamine (Sph). Optionally the amino acids histidine and alanine were inserted into the oligolysine backbone to assess a possible buffer or spacer effect. After the evaluation of biophysical properties, the best performing oligomers, containing the Stp or Sph building blocks, were compared to corresponding linear oligomers where Stp or Sph are directly integrated into the linear oligolysine row. Clear differences between the comb and linear carriers were observed in the comparison of properties such as DNA complexation ability, buffer capacity, cellular association and internalization, and gene transfer. For the Stp containing structures, the comb topology mediated an increased buffer capacity at endosomal pH. For the Sph containing structures, in sharp contrast, the linear topology displayed advantageous endosomal buffering. Interestingly, for both Stp and Sph carriers, the comb in comparison to the linear topologies mediated a higher overall cellular uptake despite a lower cell association. For Stp combs, the combined advantage in both buffering and cellular uptake resulted in a strong (10- to >100-fold) increase in DNA transfection efficiency. In the case of Sph carriers, comb topology mediated only moderately (maximum 4-fold) enhanced gene transfer over the linear topology.