Matching Items (3)

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Iterative Size Reduction of Bead Placement in Nanosphere Lithography

Description

Nanosphere lithography is a high throughput procedure that has important implications
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

Nanosphere lithography is a high throughput procedure that has important implications
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.

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Date Created
2020-05

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Algorithmic Prediction of Binding Sites of TNFα/TNFR2 and PD-1/PD-L1

Description

Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be

Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.

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Date Created
2018-05

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Thermodynamics and kinetics of DNA nanostructure assembly

Description

ABSTRACT The unique structural features of deoxyribonucleic acid (DNA) that are of considerable biological interest also make it a valuable engineering material. Perhaps the most useful property of DNA for molecular engineering is its ability to self-assemble into predictable, double

ABSTRACT The unique structural features of deoxyribonucleic acid (DNA) that are of considerable biological interest also make it a valuable engineering material. Perhaps the most useful property of DNA for molecular engineering is its ability to self-assemble into predictable, double helical secondary structures. These interactions are exploited to design a variety of DNA nanostructures, which can be organized into both discrete and periodic structures. This dissertation focuses on studying the dynamic behavior of DNA nanostructure recognition processes. The thermodynamics and kinetics of nanostructure binding are evaluated, with the intention of improving our ability to understand and control their assembly. Presented here are a series of studies toward this goal. First, multi-helical DNA nanostructures were used to investigate how the valency and arrangement of the connections between DNA nanostructures affect super-structure formation. The study revealed that both the number and the relative position of connections play a significant role in the stability of the final assembly. Next, several DNA nanostructures were designed to gain insight into how small changes to the nanostructure scaffolds, intended to vary their conformational flexibility, would affect their association equilibrium. This approach yielded quantitative information about the roles of enthalpy and entropy in the affinity of polyvalent DNA nanostructure interactions, which exhibit an intriguing compensating effect. Finally, a multi-helical DNA nanostructure was used as a model `chip' for the detection of a single stranded DNA target. The results revealed that the rate constant of hybridization is strongly dominated by a rate-limiting nucleation step.

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2011