(2fx) DNA Origami Assemblies for Reconfiguration, Actuation, and Education Modules

Over the last few decades DNA has garnered interest as a material for bottom-up- self-assembly of new devices or materials for various biological, mechanical, and chemical applications. DNA nanotechnology, specifically DNA origami, provides a platform that leverages Watson-Crick complementary base-pairing to design nanoscale devices with a pre-defined geometry capable of precise motion and controlled mechanical properties. This approach provides a framework to design dynamic nano devices capable of higher order assembly and reconfiguration. Integrating these two areas of reconfiguration and higher-order assembly to create triggered reconfigurable DNA materials remains a challenge. This work focuses on the design, reconfiguration and actuation of higher order DNA origami assemblies using DNA strand displacement, magnetic actuation, and optical traps. We envision these assemblies could form the building blocks for materials with sensing, reconfiguration, and adaptable mechanical functions. Given the wide range of emerging applications, students could also greatly benefit from earlier exposure to DNA origami nanotechnology. I will also highlight our recent efforts to introduce DNA origami education modules developing streamlined fabrication and analysis protocols that reduce time and material costs of traditional DNA origami assembly, purification, and visualization, enabling implementation in undergraduate, high school, or middle school science classrooms.

The reconfiguration and actuation of higher order assemblies remains quite limited to single device scales with multiple inputs necessary for actuation. The most common approach to actuate reconfiguration in these devices is toe-hold mediated strand displacement, which relies on the formation or dissociation of specific DNA interactions that control the device conformation. While strand displacement is a robust method of actuation, limitations include the slow response time (minutes or greater) and the need to introduce strands into solution. To overcome these limitations recent efforts have established methods that leverage externally applied fields to manipulate structures. For example, recent efforts developed an approach to integrate magnetic microbeads for direct real-time manipulation of devices with sub-second time scales, tunable applied forces, and precise spatial resolution. However, actuating DNA devices with external fields has been limited to simple systems such as levers and hinges. We designed two DNA origami assemblies in the custom software MagicDNA, which enables the design of complex 3D assemblies on the order of minutes. Here, we present two reconfigurable higher order DO assemblies: 1) The 6-bar DO assembly demonstrates reconfiguration into many different conformations using DNA strand displacement and 2) the lattice DO assembly to demonstrate rapid actuation with magnetic fields and optical tweezers. The reconfiguration and actuation of higher order DO assemblies provides tools for a variety of applications in the biological, chemical, and mechanical spaces, for example multiplexing the control of nanomachines or biomolecular interactions.

Given the interdisciplinary nature and the wide range of emerging applications of DNA origami, a variety of students could benefit from exposure to basic knowledge and methods of DNA nanotechnology. However, the cost of materials and equipment and complexity of design and characterization of nanostructures limits DNA origami experiments mainly to research focused institutions in graduate level laboratories with prior expertise and well-equipped laboratories. This work seeks to overcome crucial barriers (i.e. resources and time) to translating DNA origami methods to educational and non-graduate laboratories such as middle-school and high school science classes and primarily undergraduate institutions. The equipment needed ranges from possibly available (e.g. gel electrophoresis), to unlikely available (e.g. thermocyclers), or impractical (e.g. electron microscopy) for ready access in educational settings. Furthermore, DNA origami fabrication may take several hours to prepare and up to days for thermal annealing, and typical first step analysis methods like gel electrophoresis typically requires several hours to setup and run. In addition to these barriers the complexity of the design and fabrication process are challenging to educational translation. We developed a streamlined protocol for fabrication and analysis of static (first module) and dynamic (second module) DNA origami nanostructures that can be conducted within a 2-hour laboratory course using low-cost equipment that relies on inexpensive equipment and supplies, much of which are readily available in educational laboratories and science classrooms. The first educational experiment module focuses on a static DNA origami nanorod structure that was previously developed for drug delivery applications. The second educational experiment module focuses on a nanomechanical DNA origami structure initially designed to provide a framework and model for deformable, or compliant, nanostructures. The modules include learning tools related to DNA nanotechnology such as charge screening, mechanical deformations, conformational dynamics and free energy landscapes, nanoscale stimulus response, polymerization, and algorithmic design and assembly. The modules provide topics that relate to a variety of subjects including engineering, chemistry, physics, biology, materials science, medicine, and computer science, and hence could be worked into a range of curricula. Over the long-term, introducing a broad range of students to DNA origami would also have the potential to advance the field due to increased interest and involvement by young students, who may then pursue education, research, or career paths related to DNA nanotechnology.