A form of nanoscale steganography exists described as DNA origami cryptography which is a technique of secure information encryption through scaffold, staple, and varying docking strand self- assembling mixtures. The all-DNA steganography based origami was imaged through high-speed DNA-PAINT super-resolution imaging which uses periodic docking sequences to eliminate the need for protein binding. The purpose of this research was to improve upon the DNA origami cryptography protocol by encrypting information in 2D Rothemund Rectangular DNA Origami (RRO) and 3D cuboctahedron DNA origami as a platform of self-assembling DNA nanostructures to increase the routing possibilities of the scaffold. The initial focus of the work was increasing the incorporation efficiency of all individual docking spots for full 20nm grid RRO pattern readout. Due to this procedural optimization was pursued by altering annealing cycle length, centrifugal spin rates for purification, and lengthening docking strands vs. imager poly T linkers. A 14nm grid was explored as an intermediate prior to the 10nm grid for comparison of optimized experimental procedure for a higher density encryption pattern option. Imager concentration was discovered to be a vital determining factor in effectively resolving the 10nm grids due to high concentrations of imager strands inducing simultaneous blinking of adjacent docking strands to be more likely causing the 10nm grids to not be resolved. A 2 redundancy and 3 redundancy encryption scheme was developed for the 10nm grid RRO to be encrypted with. Further experimentation was completed to resolve full 10nm DNA-origami grids and encrypt with the message ”ASU”. The message was successfully encrypted and resolved through the high density 10nm grid with 2 and 3 redundancy patterns. A cuboctahedron 3D origami was explored with DNA-PAINT techniques as well resulting in successful resolution of the z-axis through variation of biotin linker length and calibration file. Positive results for short message ”0407” encryption of the cuboctahedron were achieved. Data encryption in DNA origami is further being explored and could be an optimal solution for higher density data storage with greater longevity of media.
The purpose of this experiment is to deliver DNA origami barrels loaded with Cas13d-gRNA binary complexes to HPV-16 and HPV-18 cervical cancer lines to make the cancer mortal. The production of Cas 13d has proven successful with a concentration of ~ 1mg/mL, but the activity assay performed has not shown conclusive evidence of Cas13d and guide RNA binary complex formation or activity. Successful annealing of the DNA origami barrel has been quantified by an agarose gel, but further quantification by TEM is in progress. Overall, steady progress is being made towards the goal of targeting HPV16 E6/E7 pre-mRNA with CRISPR/Cas13d.
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.
At ~100-nm, a DNA origami macromolecule represents one such bridge, acting as a breadboard for the decoration of single molecules with 3-5 nm resolution. It relies on the programmed self-assembly of a long, scaffold strand into arbitrary 2D or 3D structures guided via approximately two hundred, short, staple strands. Once synthesized, this nanostructure falls in the spatial manipulation regime of a nanofabrication tool such as electron-beam lithography (EBL), facilitating its high efficiency immobilization in predetermined binding sites on an experimentally relevant substrate. This placement technology, however, is expensive and requires specialized training, thereby limiting accessibility.
The work described here introduces a method for bench-top, cleanroom/lithography-free, DNA origami placement in meso-to-macro-scale grids using tunable colloidal nanosphere masks, and organosilane-based surface chemistry modification. Bench-top DNA origami placement is the first demonstration of its kind which facilitates precision placement of single molecules with high efficiency in diffraction-limited sites at a cost of $1/chip. The comprehensive characterization of this technique, and its application as a robust platform for high-throughput biophysics and digital counting of biomarkers through enzyme-free amplification are elucidated here. Furthermore, this technique can serve as a template for the bottom-up fabrication of invaluable biophysical tools such as zero mode waveguides, making them significantly cheaper and more accessible to the scientific community. This platform has the potential to democratize high-throughput single molecule experiments in laboratories worldwide.