Magic of Architecting Oligo‐DNAs: 3D Structure‐Dependent Stability and Programmable Specificity to Tumor Cells

Abstract Constructing oligo DNAs into assemblies dramatically changes the nature of those biomacromolecules. DNA crossover is one of the classic oligo‐based nanostructures in which two or more DNA double helixes connect to each other by crossover points showing the potential to be used as structural units to develop multifunctional systems. However, research about this motif is scarce, especially in the field of biomedicine. In this study, DNase‐resistant and tumor‐distinguishing DNA nano‐assemblies based on the crossover motifs called double crossover motif (DX) and three‐helix bundle motif (3HB) are constructed. Owning to the peculiarity of secondary structure, the crossover motif shows great resistance to DNases. Such resistance is closely relevant to the 3D structure, which is confirmed by circular dichroism spectra (CD) and kinetic studies. The aptamer, AS1411, is conjugated to DNA motifs on the predetermined sites with the specific number. Depending on computer‐aided design, up to two AS1411 are conjugated to DX (DX‐x), and up to three are conjugated to 3HB (3HB‐x). Surprisingly, 3HB equipped with 3 aptamers show the best tumor discriminating ability. Confirmed by super‐resolution microscopy and live cell imaging, the manipulatable tumor specificity renders the DNA motif a potential agent for biomedical applications, including in vivo application.