Expanding the DNA Nanotechnology Toolbox: Hierarchical Assembly and Self‐Sorting of DNA Tiles With Precisely Controlled Hydrophobic Behavior

ABSTRACT DNA self‐assembly is a powerful strategy to build complex nanostructures, but the chemical uniformity of natural nucleotides limits architectural diversity and long‐range organization. Here, we expand the DNA nanotechnology toolbox by integrating tunable hydrophobic effects directly into DNA double‐crossover (DX) tiles using non‐natural nucleotides. Sequence‐defined, monodisperse amphiphiles are constructed on a DNA synthesizer, with hydrophobic strength precisely controlled by the number of incorporated hexaethylene (C 12 ) units. By varying hydrophobic strength and applying thermal annealing, we define an energetic threshold that dictates the hierarchical order of hydrophobic assembly versus DNA hybridization. Weak hydrophobic effects allow base pairing to occur first, followed by hydrophobic assembly, yielding quantized DX‐tile star architectures. Strong hydrophobicity instead drives early micelle formation, followed by DNA‐mediated cross‐linking into robust spherical nucleic acids (SNAs). These crosslinked SNAs show enhanced serum stability and remain fully reversible via strand‐displacement‐triggered decrosslinking. Furthermore, DX‐tiles with identical DNA sequences but different hydrophobic segment lengths undergo narcissistic self‐sorting, assembling in one pot into distinct DX‐star and SNA populations that complement the social self‐sorting of base pairing. Finally, we demonstrate that this design principle extends to other DNA nanostructures, including three‐point star tiles, enabling cooperative, protein‐like assembly pathways and emergent functions inaccessible to DNA alone.