Fabrication of multi-layered DNA nanostructures using single-strand and double-crossover tile connectors

Abstract

DNA is an excellent and extraordinarily versatile building block that can be used to construct nanoscale objects and arrays of increasing complexity, and as a result, a considerable amount of progress has been made in DNA-directed molecular self-assembly. Here, we demonstrate the sequential fabrication of three-dimensional multi-layered DNA nanostructures by utilizing single-strand and double-crossover tile (DX) designs via substrate-assisted growth and multi-step annealing methods. We used both layering and connector tiles to synthesize the base layer for both the single strand-based and DX tile-based designs. Layering without and with connector tiles was used to produce double-layer and multi-layer designs for single strand-based designs, but only layering tiles were used for the DX tile-based design. Connector tiles provided appropriate sticky-end sets to form the designed lattice structures. Atomic force microscopy revealed that the spacing between the tiles was in good agreement with the design scheme, but the heights of the multi-layered nanostructures were found to be slightly lower than expected due to suppression by the substrate. This kind of step-wise multi-layer assembly may produce a variety of spacings to incorporate different guest molecules or aid the attachment of various types of biomolecules and nanomaterials in parallel arrays along the layers.