Browsing by Author "LaBean, Thomas H"

Biological systems have inspired interest in developing artificial molecular self-assembly techniques that imitate nature's ability to harness chemical forces to specifically position atoms within intricate assemblies. Of the biomolecules used to mimic nature's abilities, nucleic acids have gained special attention. Specifically, deoxyribonucleic acid is a stable molecule with a readily accessible code that exhibits predictable and programmable intermolecular interactions. These properties are exploited in the revolutionary structural DNA nanotechnology method known as scaffolded DNA origami. For DNA origami to establish itself as a widely used method for creating self-assembling, complex, functional materials, current limitations need to be overcome and new methods need to be established to move forward with developing structures for diverse applications in many fields. The limitations discussed in this dissertation include 1) pushing the scale of well-formed, fully-addressable origami to two and seven times the size of conventional origami, 2) testing cost-effective staple strand synthesis methods for producing pools of oligos for a specified origami, and 3) engineering mechanical properties using non-natural nucleotides in DNA assemblies. After accomplishing the above, we're able to design complex DNA origami structures that incorporate many of the current developments in the field into a useful material with applicability in wide-ranging fields, namely cell biology and photonics.

Promising applications in the design of various biological systems hold critical implications as heralded in the rising field of synthetic biology. But, to achieve these goals, the ability to synthesize in situ DNA constructs of any size or sequence rapidly, accurately and economically is crucial. Today, the process of DNA oligonucleotide synthesis has been automated but the overall development of gene and genome synthesis technology has far lagged behind that of gene and genome sequencing. This has meant that numerous ideas go unfulfilled due to scale, cost and impediments in the quality of DNA due to synthesis errors.

This thesis presents the development of a multi-tool ensemble platform targeted at gene synthesis. An inkjet oligonucleotide synthesizer is constructed to synthesize DNA microarrays onto silica functionalized cylic olefin copolymer substrates. The arrays are married to microfluidic wells that provide a chamber to for enzymatic amplification and assembly of the DNA from the microarrays into a larger construct. Harvested product is then amplified off-chip and error corrected using a mismatch endonuclease-based reaction. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents and parts that are cheaper than optical and electrochemical systems. Genes sized 160 bp to 993 bp were successfully harvested and, after error correction, achieved up to 94% of intended functionality.

Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling.

The predictable Watson Crick base pairing allows for designing selfassembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures.

To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization.

The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity.

The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by amide bonds. The solid phase synthesis of PNA allows for convenient extension of the backbone into a peptide segment enabling peptide functionalization of DNA nanostructures. We have investigated how the neutral character of PNA alters the incorporation in DNA based nanostructures compared to a DNA control using biotinylation and AFM.

Results indicate that PNA can successfully be used as a way of functionalizing DNA nanostructures. Additionally we have shown that functionalized nanostructures are capable of sensitizing cells to TGF-beta stimulation.

Synthetic biology is an emerging technology field whose goal is to use biology as a substrate for engineering. Self-assembly is one of the many methods for fabricating such synthetic biosystems.

Self-assembly is a process where components spontaneously arrange themselves into organized aggregates by the selective affinity of substructures. DNA is an excellent candidate for making synthetic biological systems using self-assembly because of its modular structure and simple chemistry. This thesis describes several

techniques which use DNA as a nano-construction material and

explores the computational capabilities of DNA self-assembly.

For this dissertation, I set out to build a biomolecular computing device with several

useful properties, including compactness, robustness, high degrees of complexity, flexibility, scalability and easily characterized yields

and convergence rates. However, a unified device that satisfies all these properties is still many years away. Instead, this thesis presents designs, simulations,

and experimental results for several distinct DNA nano-systems, as

well as experimental protocols, each of which satisfies a subset of the above-mentioned properties. The hope is that the lessons learned from building all these biomolecular computational devices would bring us closer to our ultimate goal and would eventually pave the path for a computing device that satisfies all the properties. We experimentally demonstrate how we can reduce errors in tiling assembly using an optimized set of physical parameters. We propose a novel DNA tile design

which enforces directionality of growth, reducing assembly errors. We build simulation models to characterize damage in fragile nanostructures under the impact of various external forces. Furthermore, we investigate reversible computation as a means to provide self-repairability to such damaged structures.

We suggest two modifications of an existing DNA computing device,

called Whiplash PCR machine, which allow it to operate robustly outside of controlled laboratory conditions and allow it to implement a simple form of inter-device communication. We present analysis techniques which characterize the yields and time convergence of self-assembled DNA nanostructures. We also present an experimental demonstration of a novel DNA nanostructure which is capable of tiling the plane and could prove to be a way of building 3D DNA assemblies.

In addition to being the natural genetic information carrier, DNA can also serve as a versatile material for construction of nanoscale objects. By using the base-pairing properties of DNA, we have been able to mass-produce nano-scale structures in a variety of different shapes, upon which patterns of other molecules can be further specified. The diversity of molecules and materials that can be attached to DNA and the capability of providing precise spatial positioning considerably enhance the attractiveness of DNA for nano-scale construction. A further challenge remains to use these DNA based structures for biomedical applications.

As proof-of-concept, a DNA-based nanodevice for multivalent thrombolytic delivery is designed, which intends to employ DNA nanostructures as carriers for the delivery of tissue plasminogen activator (tPA) and plasminogen. Universal modular adapter molecules that can simultaneously bind "down" to the DNA structures and "up" to these thrombolytic drugs are further proposed. We begin with exploring the molecular recognition properties provided by biotin-avidin and aptamer-ligand pairs, and are able to achieve site-specific display of certain protein targets along the DNA nanostructure scaffold. Yet for both of these approaches, only biotinylated or specially selected proteins can be patterned. We further propose to develop single-chain diabodies (scDb) as the adapter molecules. This scDb approach is highly modular and can be extended to assemble virtually any proteins and therapeutic molecules of interests, which at the same time will greatly enhance our molecular toolbox for nanoscale construction.