Barrett, The Honors College Thesis/Creative Project Collection

DNA nanotechnology is ideally suited for numerous applications from the crystallization and solution of macromolecular structures to the targeted delivery of therapeutic molecules. The foundational goal of structural DNA nanotechnology was the development of a lattice to host proteins for crystal structure solution. To further progress towards this goal, 36 unique four-armed DNA junctions were designed and crystallized for eventual solution of their 3D structures. While most of these junctions produced macroscale crystals which diffracted successfully, several prevented crystallization. Previous results used a fixed isomer and subsequent investigations adopted an alternate isomer to investigate the impact of these small sequence changes on the stability and structural properties of these crystals. DNA nanotechnology has also shown promise for a variety biomedical applications. In particular, DNA origami has been demonstrated as a promising tool for targeted and efficient delivery of drugs and vaccines due to their programmability and addressability to suit a variety of therapeutic cargo and biological functions. To this end, a previously designed DNA barrel nanostructure with a unique multimerizable pegboard architecture has been constructed and characterized via TEM for later evaluation of its stability under biological conditions for use in the targeted delivery of cargo, including CRISPR-containing adeno-associated viruses (AAVs) and mRNA.

Molecular engineering is an emerging field that aims to create functional devices for modular purposes, particularly bottom-up design of nano-assemblies using mechanical and chemical methods to perform complex tasks. In this study, we present a novel method for constructing an RNA clamp using circularized RNA and a broccoli aptamer for fluorescence sensing. By designing a circular RNA with the broccoli aptamer and a complementary DNA strand, we created a molecular clamp that can stabilize the aptamer. The broccoli aptamer displays enhanced fluorescence when bound to its ligand, DFHBI-1T. Upon induction with this small molecule, the clamp can exhibit or destroy fluorescence. We demonstrated that we could control the fluorescence of the RNA clamp by introducing different complementary DNA strands, which regulate the level of fluorescence. Additionally, we designed allosteric control by introducing new DNA strands, making the system reversible. We explored the use of mechanical tension to regulate RNA function by attaching a spring-like activity through the RNA clamp to two points on the RNA surface. By adjusting the stiffness of the spring, we could control the tension between the two points and induce reversible conformational changes, effectively turning RNA function on and off. Our approach offers a simple and versatile method for creating RNA clamps with various applications, including RNA detection, regulation, and future nanodevice design. Our findings highlight the crucial role of mechanical forces in regulating RNA function, paving the way for developing new strategies for RNA manipulation, and potentially advancing molecular engineering. Although the current work is ongoing, we provide current progress of both theoretical and experimental calculations based on our findings.