Self-Assembly Of DNA Nanostructures And Its Application In DNA Detection

As the basis of living systems, the specificity of DNA base-paring has been discovered by Watson and Crick for over 60 years. The precisely complementary pairing of adenine (A) with thymine (T) and guanine (G) with cytosine (C), which is the most significant property of DNA, allows the achievements of specific molecular recognition. This property makes DNA a unique molecule and promotes the development of gene engineering dramatically. In addition, the fact that DNA molecules with arbitrary sequences have been commercially available enables the ability to produce novel DNA molecules according to the Watson-Crick base paring. Therefore, it is possible to design molecules with topologies using DNA as building blocks.Since N. C. Seeman first proposed the concept of structural DNA nanotechnology in 1982, this field has delivered numerous advances. DNA nanotechnology is a bottom-up self-assembled fabrication method which uses DNA as building blocks to construct complex and addressable nanostructures in one-(1D), two-(2D) and three-dimensions (3D). A mile stone in this field was the introduction of DNA origami by Rothemund in 2006. A long single stranded DNA served as a scaffold was folded with the help of hundreds of short single stranded DNA called staple strands to form 2D nanostructures. Following that, a series of 3D nanostructures were constructed by DNA origami. Furthermore, by using short single stranded DNA as tiles and bricks without a scaffold strand, Yin fabricated various 2D and 3D complex nanostructures. The remarkable feature of this strategy is that arbitrary structures can be sculpted from the designed large-scale DNA canvas so that no new design is required for each structure. As the rapid development of DNA nanotechnology, its applications have also been exploited greatly, especially in single-molecule detection, material organization and dynamic systems, and it holds great potential for future scientific and technological fields.This thesis describes a multiplexed DNA detection strategy by positional encoding/decoding based on self-assembled DNA nanostructures (PED-SADNA). PED-SADNA allows the simultaneous achievement of facile positional encoding/decoding and fast hybridization kinetics in a solution assay format. In addition, a strategy to assemble DNA polygonal cavities with tunable shapes and sizes have been demonstrated, in which a new angle control principle is developed. The specific research works are as follows:1. Research on multiplexed DNA detection based on positional encoding/decoding with self-assembled DNA nanostructures. A self-assembled three-dimensional core DNA structure with a registry marker asymmetrically positioned at one end and multiple types of target-binding capture probe sequences placed at regular intervals (CU) is fabricated for the unambiguous positional encoding of DNA target information. Accordingly, multiple satellite DNA structures containing detection probe sequences (DU) for the remaining portions of corresponding targets are individually assembled. The presence of each target will direct the respective DU to the partner site of CU through hybridization. Staining of DNA nanostructures with uranyl formate and visualization with transmission electron microscopy (TEM) allow positional decoding and unambiguous identification of target DNA.2. Research on construction of DNA polygonal cavities with tunable shapes and sizes. A monomer with controlled slope on one end, enabled by altering the helical length gap, is fabricated to hierarchically construct shape- and size-tunable polygonal cavities. Sticky ends anchored on the tail (Sticky End 1) of the monomer are complementary to the sticky ends on the internal surface (Sticky End 2). The hybridization of the sticky ends between the monomers enables the construction of polygonal cavities. In addition, the cavity size, with the side length corresponding to the distance between Sticky End 2 and the tail of the monomer, can be readily controlled by changing the location of Sticky End 2 along the helical axis, with Sticky End 1 immobilized.