UC San Diego

DNA nanotechnology as a versatile tool has been growing rapidly in the past three decades, with applications across different disciplines such as drug delivery, imaging, and even computing. Its versatility stems from the programmable nature of DNA and the ability to self-assemble through the A-T and C-G base pairing by hydrogen bonds. These features make DNA nanotechnology the ideal tool for mimicking and studying biological processes that are hard to otherwise model. In this work, the process we focus on is Clathrin-Mediated Endocytosis (CME). CME is one of the major mechanisms for cell entry. This pathway internalizes Influenza A, vesicular stomatitis virus, and many others. Mimicking this process will help us further understand it, and also find ways to utilize it in the field of drug delivery.The process of CME can be described in 3 major steps. The first step is array formation. Clathrin can cluster on cell surface in the form of triskelion, to form small patches of an array. The second step involves the recruitment of adaptor proteins, that transforms the array into three-dimensional lattices to encapsulate the foreign agent. The third step is the transportation of the vehicle inside the cell, along with the disassembly of the vehicle to release the agent. Specifically for each stage, we designed mimicking mechanisms using DNA nano-structures. For the array stage, we designed a three-point-star motif to mimic the triskelion structure, and functionalized it with cholesterol to integrate the array to the cell membrane. Characterization through liquid atomic force microscopy (AFM) showed clear hexagonal pattern, and in vitro cell experiment also showed the integration of the arrays to cell membranes. For the transition stage, we designed a reversible 2D-3D transition mechanism that allowed the 2D arrays to transform into 3D particles with the addition of a particular stimuli, that is a DNA single strand. The transformability and reversibility were confirmed through polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and AFM. For the delivery stage, we designed several 3D DNA structures for better drug delivery efficiency, and the designs were also characterized by PAGE and AFM. The successful designs for all three stages led us closer to understanding the structural transformation of clathrin triskelion during CME. The 2D-3D transition mechanism also has the potential to be used in other systems, such as stimuli-controlled drug release and DNA computing using single strand DNA.