The large research effort focused on enhancing nonviral transfection vectors has clearly demonstrated that their macromolecular structure has a significant effect on their transfection efficacy. The 3D branched polymeric structures, such as dendrimers, have proved to be a very effective structure for polymeric transfection vectors; however, so far the dendritic polymers have not delivered on their promise. This is largely because a wide range of dendritic polymer materials with tailored multifunctional properties and biocompatibility required for such applications are not yet accessible by current routes. Herein, we report the design and synthesis of new 3D "Single Cyclized" polymeric gene vectors with well-defined compositions and functionalities via a one-step synthesis from readily available vinyl monomers. We observe that this polymer structure of a single chain linked to itself interacts differently with plasmid DNA compared to conventional vectors and when tested over a range of cell types, has a superior transfection profile in terms of both luciferase transfection capability and preservation of cell viability. This new knotted structure shows high potential for gene delivery applications through a combination of simplicity in synthesis, scalability, and high performance.