Sequence-defined oligoaminoamides (OAAs) provide a promising option for non-viral nucleic acid delivery and effective gene therapy. Solid phase-assisted synthesis enables the design of carriers with different structural motifs. Five nucleic acid-binding carriers with a precise 4-arm topology were designed, all containing lysines for branching, the cationizable artificial amino acid succinoyl-tetraethylene-pentamine (Stp) and histidines, together promoting endosomal escape, cysteines for stabilization by disulfide cross-linking, and N–terminal azidolysines displaying possible anchor points for shielding/targeting. The structures differ in additional (i) tyrosines or tryptophanes as block-wise tripeptides (Y₃, W₃) or (ii) alternating motifs with the Stp and histidines (Stp-H-W) to stabilize polyplexes by hydrophobic interactions, or (iii) N–terminal leucines (L) as hydrophobic end groups. The impact of the different structures on transfection of plasmid DNA (pDNA) in N2A and HUH7 tumor cells and DC2.4 dendritic cells was evaluated. Small interfering RNA (siRNA) was tested in N2A-eGFPLuc cells. In all cell lines, the tyrosine tripeptide incorporation led to high cytotoxicity, and the tryptophane tripeptide motif, though well-tolerated, did not improve transfection. Interestingly, for the HUH7 hepatoma and DC2.4 dendritic cells, but not the N2A neuroblastoma cells, the (Stp-H-W) motif, and the terminal leucine (L) motif mediated the best transfection results. These two 4-arm structures may represent suitable carriers for DNA vaccination.