Expanding the Toolbox for DNA Nanotechnology

DNA consists of four nucleobases adenine (A), cytosine (C), guanine (G) and thymine (T), and hydrogen bonds could be formed between complimentary nucleobases. DNA Nanotechnology is mainly based on complementarity of DNA sequences. Chapter 1 is a brief introduction of method development, applications, and assembly breakthroughs of DNA nanostructures. Chapter 2 to Chapter 5 are my research summaries about new strategies and applications of DNA nanostructures assembly. In Chapter 2, we observed that strand displacement could occur in the context of DNA triplexes. However, the rate of strand displacement process in triplex is slower than that in duplex. In this study, there are two molecular systems, and one contains a duplex toehold and the other one contains a triplex toehold. The strand displacement in two molecular systems was tested by native polyacrylamide gel electrophoresis. This study provides a new strategy for sequence-specific strand displacement and it is useful for the construction of dynamic DNA nanostructures and control of smart DNA nanodevices. In Chapter 3, we discovered a class of unusual DNA duplexes, which were assembled by polythymines (polyT) in the present of melamine (MA). The formation of such DNA structures relies on extensive hydrogen-bonding between T and MA. Each T forms three H-bonds with a MA molecule and each MA simultaneously forms H-bonds with two Ts. The resulting duplexes have been characterized by native polyacrylamide gel electrophoresis, UV spectrophotometer, and circular dichroism spectrophotometer. This study not only diversifies DNA nucleobases, but also provides a potentially useful tool for engineering DNA nanostructures. In Chapter 4, we proposed a strategy to increase stability of DNA nanostructures. DNA triplexes were successfully added onto each edge of a DNA tetrahedron and the newly formed tetrahedra were more stable either under physical force or high temperature. The triplex-forming tetrahedra were confirmed by native polyacrylamide gel electrophoresis and atomic force microscope. This study not only stabilizes DNA nanostructures, but also provides possible attachment for DNA nanostructures. In Chapter 5, we applied DNA nanostructures as delivery of class-P unmethylated cytosine-phosphate-guanosine (CpG) oligodeoxynucleotide (ODN). These large DNA nanostructures could protect the CpG ODN with phosphodiester (PO) backbone from DNase, and thus enhance efficiency of CpG ODN. These structures were characterized by native polyacrylamide gel electrophoresis and atomic force microscopy. Cytotoxicity of the DNA structures was tested by MTT assay, and taken-up amount of Cy3 labeled DNA nanostructures by cells was tested by flow cytometry and light microscope, and cytokine (TNF-α) released in cells after incubation with CpG ODN was tested by ELISA. This study provides a simple assembly strategy to protect DNA structures from degradation in cells, which make it efficient in treatment of cancer and infectious diseases