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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.
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.
for facile, low cost scaling of nanostructures. However, current benchtop experiments have
limitations based on the placement of molecular species that exhibit greater than singlemolecular binding. In addition, reliance upon bottom-up self-assembly of close-packed
nanospheres makes it problematic to resolve images using low-cost light microscopes due to the
spacing limitations smaller in magnitude than light wavelength. One method that is created to
resolve this issue is iterative size reduction (ISR), where repetitive ‘iterative’ processes are
employed in order to increase the precision at which single molecules bind to a given substrate.
ISR enables inherent separation of nanospheres and therefore any subsequent single molecule
binding platforms. In addition, ISR targets and encourages single-molecule binding by
systematically reducing binding site size. Results obtained pursuing iteratively reduced
nanostructures showed that many factors are needed to be taken into consideration, including
functionalization of nanosphere particles, zeta potential, and protonation-buffer reactions.
Modalities used for observation of nanoscale patterning and single-molecule binding included
atomic force microscopy (AFM) and ONI super-resolution and fluorescence microscopy. ISR
was also used in conjunction with zero mode waveguides, which are nanoapertures enabling realtime single molecule observation at zeptoliter volumes. Although current limitations and
obstacles still exist with reproducibility and scalability of ISR, it nonetheless exhibits limitless
potential and flexibility in nanotechnology applications.