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This thesis will focus on the computational design of structural and dynamic nucleic acid systems, particularly for new type of DNA structure design and high precision control of gene expression in vivo. Firstly, a new type of fundamental DNA structural motif, the layered-crossover motif, will be introduced. The layered-crossover allow non-parallel alignment of DNA helices with precisely controlled angle. By using the layered-crossover motif, the scaffold can go through the 3D framework DNA origami structures. The properties of precise angle control of the layered-crossover tiles can also be used to assemble 2D and 3D crystals. One the dynamic control part, a de-novo-designed riboregulator is developed that can recognize single nucleotide variation. The riboregulators can also be used to develop paper-based diagnostic devices.
DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build nanodevices ranging from in vitro small molecule biosensing to sophisticated in vivo therapeutic drug delivery systems and multi-enzyme networks.
This thesis focuses on how to use DNA nanostructures as programmable biomolecular scaffolds to arranges enzymatic systems. Presented here are a series of studies toward this goal. First, we survey approaches used to generate protein-DNA conjugates and the use of structural DNA nanotechnology to engineer rationally designed nanostructures. Second, novel strategies for positioning enzymes on DNA nanoscaffolds has been developed and optimized, including site-specific/ non site-specific protein-DNA conjugation, purification and characterization. Third, an artificial swinging arm enzyme-DNA complex has been developed to mimic substrate channeling process. Finally, we extended to build a artificial 2D multi-enzyme network.
DNA nanotechnology, the self-assembly of DNA into 2D and 3D nanoscale structures facilitated via Watson and Crick base pairing, provides alternative solutions for biomedical challenges, especially for therapeutic cargo delivery, because it is easily fabricated, exhibits low cytotoxicity, and high biocompatibility. However, the stability of these DNA nanostructures (DN) under cellular environment presents an issue due to their requirements for high salt conditions and susceptibility to nuclease degradation. Furthermore, DNs are typically trapped in endolysosomal compartments rather than the cytosol, where most of their cargo must be delivered. Many attempts to mitigate the stability issue have been made in recent years. Previously, our lab designed an endosomal escape peptide, Aurein 1.2 (denoted “EE, for endosomal escape)”, combined with a decalysine sequence (K10) proven to electrostatically adhere to and protect DNs under cell culture conditions. Unfortunately, this molecule, termed K10-EE, only resulted in endosomal escape in absence of serum due to formation of a protein corona on the surface of the coated DN.6 Therefore, we now propose to electrostatically coat the DN with a polymer composed of decalysine (K10), polyethylene glycol (PEG, which demonstrates antibiofouling properties), and peptide EE: K10- PEG1k-EE. Described herein are the attempted synthetic schemes of K10-PEG1k-EE, the successful synthesis of alternative products, K10-(EK)5 and K10-(PEG12)2-EE, and their resulting impacts on DN stability under biological conditions. Coating of the K10-(EK)5 with a DNA barrel origami demonstrated inefficient stabilizing capability in serum. Future studies include testing K10- (PEG12)2-EE protection for a variety of nucleic acid-based structures.
With climate change threatening to increase the frequency of global pandemics, the need for quick and adaptable responses to novel viruses will become paramount. DNA nanotechnology offers a highly customizable, biocompatible approach to combating novel outbreaks. For any DNA nanotechnology-based therapeutic to have future success in vivo, the structure must be able to withstand serological conditions for an extended time period. In this study, the stability of a wireframe DNA snub cube with attached nbGFP used to bind a nonessential viral epitope on Pseudorabies virus is evaluated in vitro both with and without one of two modifications designed to enhance stability: 1) the use of trivalent spermidine cations during thermal annealing of the nanostructure, and 2) the introduction of a polylysine-polyethylene glycol coating to the conjugated nanostructure. The design, synthesis, and purification of the multivalent inhibitor were also evaluated and optimized. Without modification, the snub cube nanostructure was stable for up to 8 hours in culture media supplemented with 10% FBS. The spermidine-annealed nanostructures demonstrated lesser degrees of stability and greater degradation than the unmodified structures, whereas the polylysine-coated structures demonstrated equivalent stability at lower valencies and enhanced stability at the highest valency of the snub cube inhibitor. These results support the potential for the polylysine-polyethylene glycol coating as a potential method for enhancing the stability of the snub cube for future in vivo applications.