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Using Modular Preformed DNA Origami Building Blocks to Fold Dynamic 3D Structures

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Using Modular Preformed DNA Origami Building Blocks to Fold Dynamic 3D Structures Using Modular Preformed DNA Origami Building Blocks to Fold Dynamic 3D Structures Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Gunter Eickert, B.S. Graduate Program in Mechanical Engineering The Ohio State University 2014 Thesis Committee: Carlos Castro, Advisor Haijun Su Copyright by Gunter Erick Eickert 2014 Abstract DNA origami is a bottom-up approach that takes advantage of DNA’s structure and lock key sequencing to build nano scale machines and structures. By introducing specific single stranded DNA (ssDNA) “staples”, a ssDNA “scaffold” can be folded into a desired 2D or 3D structure. Using this method, a variety of shapes have been formed, including tetrahedrons and octahedrons. These structures have been explored as drug carriers, enzyme platforms, signal markers, and for other medical and research uses. However, each of these structures must be specifically designed and often pertain to only one particular application. A DNA origami structure that functions as a building block was designed to allow for the creation of several different 3D structures without the need for a lengthy design process. An annealing process called a thermal ramp was then used to fold the structures. The building block was made of four equilateral triangles arranged into a parallelogram. This is ideal since a parallelogram can be used to form three of the five platonic solids, in addition to many non-platonic shapes. The parallelogram was successfully folded into a tetrahedron and an octahedron by introducing staples that bound to overhangs on its edges. By utilizing strand displacement, the 3D structures were able to be unfolded back into building blocks and then refolded into new 3D shapes. All of these foldings and unfoldings were performed at room temperature, without the need for a thermal ramp. ii Each structure was verified using transmission electron microscopy. The ability to switch between a tetrahedron and an octahedron with the same parallelograms suggests that the parallelograms could also be used to form other structures, such as an icosahedron. The parallelogram building block is both modular and dynamic. These features could allow the structure to be used for carrying and releasing a drug and could make it possible to control the kinetics of enzymes attached to the DNA structure. Both of these applications can be explored since the parallelogram can take on multiple conformations and can be switched between them without the need for a complete redesign. iii Acknowledgments I would like to thank Dr. Castro for all of his advice and guidance. I would also like to thank all the students in the Nanoengineering and Biodesign Lab and the OhioMOD team for their help in my research. iv Vita 2007................................................Gibsonburg High School 2012................................................B.S. Biomedical Engineering, The Ohio State University 2014................................................M.S. Mechanical Engineering, The Ohio State University Fields of Study Major Field: Mechanical Engineering v Table of Contents Abstract............................................................................................................................................ii Acknowledgments..........................................................................................................................iv Vita....................................................................................................................................................v List of Figures................................................................................................................................vii Chapter 1: An Introduction to DNA Origami.............................................................................1 Chapter 2: Design of a Modular and Dynamic Structure.........................................................19 Chapter 3: Results...........................................................................................................................32 Citations.........................................................................................................................................47 vi List of Figures Figure 1. Structure of DNA............................................................................................................2 Figure 2. The Holliday Junction.....................................................................................................3 Figure 3. DNA Origami Cube........................................................................................................4 Figure 4. 3D Tiled DNA Origami Canvases.................................................................................5 Figure 5. 2D Tiled DNA Origami Images.....................................................................................7 Figure 6. 3D Tiled DNA Origami Canvases.................................................................................8 Figure 7.3D Tiled DNA Origami Images......................................................................................9 Figure 8. Scaffolded DNA Origami Explanation.......................................................................10 Figure 9. Modular Scaffolded DNA Origami Images................................................................10 Figure 10. Modular Scaffolded DNA Origami Using a Framework.......................................11 Figure 11. 3D Scaffolded DNA Origami Explanation...............................................................12 Figure 12. Scaffolded DNA Polymer and Icosahedron.............................................................13 Figure 13. Strand Displacement..................................................................................................14 Figure 14. Dynamic Scaffolded DNA Frame.............................................................................16 Figure 15. DNA Origami Enzyme Example Complexes...........................................................18 Figure 16. A Parallelogram and Platonic Solids it Can Form.................................................21 Figure 17. Parallelogram Scaffold Routing................................................................................22 Figure 18. caDNAno User Interface............................................................................................23 vii Figure 19. Complete Form 2 and Form 3 Parallelagram Design Schematics........................24 Figure 20. Folding a Parrallelogram into a Tetrahedron..........................................................26 Figure 21. Folding Two Parallelograms into an Octahedron...................................................27 Figure 22. Transforming a Form 2 Parallelogram into a Form 3 Parallelogram...................28 Figure 23. Strand Displacement Kinetics...................................................................................29 Figure 24. Experiment Design......................................................................................................31 Figure 25. Initial Design and Images of a Parallelogram..........................................................33 Figure 26. Folding Initial Design into Tetrahedrons and Unfolding with Heat....................34 Figure 27. Agarose Gel of Final Structures.................................................................................36 Figure 28. Images of Folding Sequence Starting with Octahedrons.......................................37 Figure 29. Images of Folding Sequence Starting with Tetrahedrons......................................38 Figure 30. How a Parallelogram can Fold onto Itself................................................................40 Figure 31. Ways to Interpret TEM Images of Tetrahedrons and Octahedrons.....................42 Figure 32. Outlined TEM Images................................................................................................43 Figure 33. How to Fold an Icosahedron.....................................................................................45 Figure 34. An Eight Sided Non-platonic Solid..........................................................................46 Figure 35. A DNA Origami Delivery System.............................................................................47 viii Chapter 1: An Introduction to DNA Origami The ability to design and build nanoscale structures and machines is an idea that has the potential to impact every aspect of our lives. Nanostructures are being explored to improve solar energy harvesting[1] and to carry drugs to more effectively combat tumors[2]. Nano- and microfabrications are created using either a top-down method (by which a structure is made as the result of removing specific material from an existing bulk material) or a bottom-up method (by which a structure is assembled from individual smaller parts). An example of the top-down method in use today is photolithography, a process used to fabricate the chips made of nanoscale structures that ushered our world into the modern era of computers. Life, on the other hand, the most abundant user of nanoscale structures and machines, uses a bottom-up approach. Both proteins and DNA, which define a large portion of our biology, are synthesized using a bottom-up method - proteins being made from amino acids and DNA being made of nucleic acids. It should come as no surprise, then, that the bottom-up assembly found in biology has been
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