We have developed novel techniques for manufacturing vesicles with predefined attachments to scaffolds of DNA, and have studied the underlying mechanism(s) of this DNA directed vesicle formation by capturing intermediates. These DNA scaffolds are self-assembled by the origami method, which can use DNA as a programmable building block to form diverse structures: two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra [1-5].
Nano-templated vesicles are prepared using rigid rings of bundled DNA. Single phosphatidyl ethanolamine (PE) lipids are coupled to these rings first by covalent conjugation with an oligonucleotide (oligo) "anti-handle", then by that oligo's sequence-specific hybridization to one of several (0, 1, 2, ..., 16) single-stranded "handles" on the DNA ring, designed to protrude from its interior. Vesicles are then formed in a solution of these ring complexes, excess phospholipid and detergent as the detergent is dialyzed away over several hours. Micelles preferentially nucleate around the alkyl chain of each PE inside the ring, and their growth during dialysis determines the volume of lipid in the final structures formed. Ring-PE lipid-vesicles bear exactly one ring per vesicle in characteristic transmission electron micrographs, with a size close to the inner diameter of its ring template.
Chapter 1 provides an overview of the significance and roles of engineering membranes in vitro. Biological membranes are incredibly complex, which in turn makes studying structure and function of membrane protein difficult in the absence of an artificial bilayer. Even more so, current limitations of producing high quality liposomes with reproducible techniques are placing more strain on elucidating the mechanisms of reconstitution. However, the emergence of the field of DNA Origami in 2006 truly revolutionized the limitless abilities to create 2D and 3D structures with function. We took advantage of this field by developing geometries to facilitate membrane growth.
Chapter 2 reports a new method for templating vesicles with a uniform size and shape using DNA origami rings bearing inner handles facing 0° to the center. DNA origami rings of varying diameters can be designed with functional handles for templating the "Saturn" structure. Once the method was established, rings of varying handle angles were synthesized to determine their effects on the final vesicle structures.
Chapter 3 explores the parameters that affect the quantity of lipids assembling inside the template. These include ultracentrifugation time, detergent to lipid ratio, and dialysis conditions. In order to elucidate the mechanism of formation of our final templated structures, we performed mechanistic studies on 60-nm rings, systematically varying the initial number of lipid molecules anchored inside each ring. The capture of crucial intermediates: circular thin lipidic membrane, lipid bilayer torus, continuous outer bilayer, and seeded small unilamellar vesicles helped us understand how the vesicles are formed.
Chapter 4 summarizes the main results of the thesis and provides future prospectives on the potential expansion of DNA origami technology. A handful of new opportunities are presented based on control in the organization of DNA materials. Taking advantage of this machinery and applying it to the central problems in engineering, biology, chemistry, physics, and medicine will allow the field to elevate to the next level with promises of becoming a vital area of research.
|School Location:||United States -- Connecticut|
|Source:||DAI-B 76/07(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Chemistry, Nanoscience|
|Keywords:||DNA nanotechnology, DNA origami, Liposomes|
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