Higher-order DNA Nanoarchitectures For Biological Applications

DNA is a kind of natural nanostring with delicate structure features and remarkablemolecular recognition properties. In the past decades, structural DNA nanotechnology hasgenerated a series of complex DNA nanostructures in different dimensions using DNA as rawmaterials, and size, shape, topological properties, chiral, periodicity of these structures have beenprecisely controlled. However, it remains a challenge to prepare higher-order DNAnanoarchitectures with large size in large scale, which is important for realizing complicatedfunctions. Another approach is to assemble preformed small structures (e.g. small tiles and evenorigamis), which called “DNA bricks”, into supramolecular assemblies of higher-order DNAnanoarchitectures. Here we constructed dendrimer-like and chain-like nanostructures with highlypurifed DNA tetrahedra and carried out some biological applications. The main findings are asfollows:(1) Self-assembly, purification and quantification of DNA tetrahedraWe introduced size exclusion chromatography (SEC) and anion exchange chromatography(SEC) to prepare highly purifed tetrahedral DNA nanocages in large scale and demonstrated thatprecise quantifcation of DNA nanocages was the key to the formation of higher-order DNAnanoarchitectures. We successfully purifed a series of DNA nanocages with diferent sizes,including seven DNA tetrahedra with diferent edge lengths (7,10,13,17,20,26,30bp) and onetrigonal bipyramid with a20-bp edge. These highly purifed and aggregation-free DNAnanocages could be self-assembled into higher-order DNA nanoarchitectures with extraordinarilyhigh yields (98%for dimer and95%for trimer). As a comparison, unpurifed DNA nanocagesresulted in low yield of14%for dimer and12%for trimer, respectively. AFM images clearlypresented the characteristic structure of monomer, dimer and trimer, implying the purifed DNAnanocages well-formed the designed nanoarchitectures. Therefore, we have demonstrated thathighly purifed DNA nanocages are excellent “bricks” for DNA nanotechnology and show greatpotential in various applications of DNA nanomaterials.(2) Dendrimer-like DNA nanostructures for anticancer drug deliveryDNA tetrahedra were designed to overhang four cohesive ends (25bases) and could furtherhybridize with other tetrahedron DNA. Dendrimer-like DNA nanostructures were preparedthrough a step-by-step strategy using DNA tetrahedron as monomers and we successfullyconstructed the third generation dendrimer-like DNA nanostructures. In addition, we compared the effects of Doxorubicin-loaded G0(DNA tetrahedron) and G1(the third generationdendrimer-like DNA nanostructures) on overcoming drug resistance in breast cancer cells.Results showed two drug delivery system could significantly inhibit the growth of thedrug-resistant cells and Doxorubicin-loaded G1was better than Doxorubicin-loaded G0.(3) DNA tetrahedron chain polymers for multicolor barcodesWe constructed tandem structure within a theoretical size (88nm) using seven DNAtetrahedra and these structures showed different forms including line-shape, L-shape and randomcoil under AFM imaging. We designed36fluorescence-based barcodes using the heptamer inwhich every tehtahedron was labeled Alexa488(green), ROX (orange) or Cy5(red).(4) Self-assembled multivalent DNA nanostructures for therapeutic Delivery ofplatelet-derived growth factor (PDGF) aptamerIncorporating PDGF aptamers into DNA tetrahedra solved the following questions: firstly,DNA tetrahedra display increased resistance to nuclease degradation. PDGF aptamer started tobe degraded after it was incubated with cell growth medium for6hours, but PDGFaptamer-DNA tetrahedron conjugates (TH20-1Apt and TH20-4Apt) were still stable after theywere incubated with cell growth medium for12hours; secondly, multivalence enhanced PDGFaptamer’s affinity with targets; thirdly, DNA tetrahedron increased intracellular uptake of PDGFaptamer; lastly, PDGF aptamer-DNA tetrahedron conjugates significantly inhibit the growth ofthe cancer cells.(5) Purification of DNA-origami nanostructures by glycerol rate-zonal centrifugationWe purified square DNA origami through glycerol rate-zonal centrifugation. In contrast tothe agarose-gel electrophoresis-based purifcation method, glycerol rate-zonal centrifugationmethod has some advantages: firstly, high-throughput recovery and simple operation.Electrophoresis is generally used to yield0.1–1mg purified DNA nanostructures, butobtaining>100mg of purified structure through electrophoresis is laborious because ofextraction, desalting and concentration steps after electrophoresis. For rate-zonal centrifugation,structures were purified at the scale of0.1–100mg per centrifuge tube and multiple centrifugetubes for each centrifugation could be used at the same time. Secondly, purified structures don’tcontain contaminants such as DNA staining reagents and agarose-gel residues.