Hierarchical assembly of metal nanoparticles, quantum dots and organic dyes using DNA origami scaffolds.

The self-assembly of nanoscale elements into three-dimensional structures with precise shapes and sizes is important in fields such as nanophotonics, metamaterials and biotechnology. Short molecular linkers have previously been used to create assemblies of nanoparticles, but the approach is limited to small interparticle distances, typically less than 10 nm. Alternatively, DNA origami can precisely organize nanoscale objects over much larger length scales. Here we show that rigid DNA origami scaffolds can be used to assemble metal nanoparticles, quantum dots and organic dyes into hierarchical nanoclusters that have a planet-satellite-type structure. The nanoclusters have a tunable stoichiometry, defined distances of 5-200 nm between components, and controllable overall sizes of up to 500 nm. We also show that the nanoscale components can be positioned along the radial DNA spacers of the nanostructures, which allows short- and long-range interactions between nanoparticles and dyes to be studied in solution. The approach could, in the future, be used to construct efficient energy funnels, complex plasmonic architectures, and porous, nanoengineered scaffolds for catalysis.

M1.3--a small scaffold for DNA origami .

The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides. Here we report the convenient preparation of a 704 nt fragment dubbed "M1.3" as a linear or cyclic scaffold and the assembly of small origami structures with just 15-24 staple strands. A typical M1.3 origami is large enough to be visualized by TEM, but small enough to show a cooperativity in its assembly and thermal denaturation that is reminiscent of oligonucleotide duplexes. Due to its medium size, M1.3 origami with globally modified staples is affordable. As a proof of principle, two origami structures with globally 5'-capped staples were prepared and were shown to give higher UV-melting points than the corresponding assembly with unmodified DNA. M1.3 has the size of a gene, not a genome, and may function as a model for gene-based nanostructures. Small origami with M1.3 as a scaffold may serve as a workbench for chemical, physical, and biological experiments.

Cellular immunostimulation by CpG-sequence-coated DNA origami structures.

To investigate the potential of DNA origami constructs as programmable and noncytotoxic immunostimulants, we tested the immune responses induced by hollow 30-helix DNA origami tubes covered with up to 62 cytosine-phosphate-guanine (CpG) sequences in freshly isolated spleen cells. Unmethylated CpG sequences that are highly specific for bacterial DNA are recognized by a specialized receptor of the innate immune system localized in the endosome, the Toll-like receptor 9 (TLR9). When incubated with oligonucleotides containing CpGs, immune cells are stimulated through TLR9 to produce and secrete cytokine mediators such as interleukin-6 (IL-6) and interleukin-12p70 (IL-12p70), a process associated with the initiation of an immune response. In our studies, the DNA origami tube built from an 8634 nt long variant of the commonly used single-stranded DNA origami scaffold M13mp18 and 227 staple oligonucleotides decorated with 62 CpG-containing oligonucleotides triggered a strong immune response, characterized by cytokine production and immune cell activation, which was entirely dependent on TLR9 stimulation. Such decorated origami tubes also triggered higher immunostimulation than equal amounts of CpG oligonucleotides associated with a standard carrier system such as Lipofectamine. In the absence of CpG oligonucleotides, cytokine production induced by the origami tubes was low and was not related to TLR9 recognition. Fluorescent microscopy revealed localization of CpG-containing DNA origami structures in the endosome. The DNA constructs showed in contrast to Lipofectamine no detectable toxicity and did not affect the viability of splenocytes. We thus demonstrate that DNA origami constructs represent a delivery system for CpG oligonucleotides that is both efficient and nontoxic.