DNAs on colloidal surfaces: visualization and quantification using super-resolution microscopy

Chapter 1: A literature review on DNA-coated colloids (DCC): synthesis, assembly, and open questions, with an introduction to various super-resolution methods for characterization Chapter 2: Streptavidin vs. Click particles and their crystallization In this chapter, we aim to understand the DNA-mediated assembly behavior of DCC functionalized via Click Chemistry (CCP) and Streptavidin-Biotin interaction (BSP). We synthesized CCPs and formed 2D and 3D crystals, while BSPs purchased from companies resulted in disordered aggregates. To understand the reason for the difference in assembly behavior, we examined potential factors such as surface DNA density, distribution, and particle surface roughness. Both systems revealed similar DNA density and surface roughness, leading us to probe the last factor, DNA distribution. To investigate the influence of DNA distribution, we visualized the surface of DCC on a nanoscale level, employing STED super-resolution microscopy, enabling us to study spatial heterogeneities in the DNA grafting density. We correlated our measurements on the nanoscopic structure of the brush to the assembly dynamics of the particles to study the interplay between spatial heterogeneities and particle crystallization. We have developed an image analysis pipeline for particles imaged via STED and quantified DNA distributions on CCPs and BSPs using statistical and autocorrelation analysis. In the STED resolution limit, BSPs demonstrate a remarkably homogeneous DNA distribution. Our investigation has ruled out DNA density, surface roughness, and DNA distribution as hindrances to BSP crystallization. Chapter 3: Further Investigating the causes of aggregation in Streptavidin-Coated Colloids In this chapter, we aimed to understand why BSPs don’t crystallize under the same conditions where CCPs do. After ruling out differences in the density and spatial distribution of the surface coating, we explore two potential factors, unique to BSP compared to CCP, that could contribute to BSP aggregation. These factors include the nanoscale variations in DNA density due to SA-multivalency and the absence of a polymer brush in BSP, unlike CCP where its presence prevents non-DNA-mediated interactions, specifically unspecific binding to particle surfaces. To eliminate multivalency as a factor, we conducted assembly experiments using monovalent BSP. Additionally, to investigate unspecific binding, we conjugated Streptavidin molecules to a model system, CCP. Despite these efforts, both experiments showed aggregation, indicating the pivotal role of Streptavidin molecule chemistry in BSP aggregation. Chapter 4: 3D DNA-PAINT on 1um particles to observe DNA distribution in lower length scales In this chapter, our goal is to map the 3D positions of individual DNA strands on colloids. We selected two contrasting systems: homogeneous CCPs and patchy CCPs. We developed a model system for patchy particles using gold spheres as blocking agents. Employing personalized DNA-PAINT, we visualized DNA density on CCPs and utilized a PSF fitting algorithm to pinpoint DNA positions on a 3D sphere representing our colloids. Our approach effectively differentiated between these two systems by identifying DNA patches of several tens of nanometers on the particles. Chapter 5: Probing the impact of an azobenzene photoswitch on DNA hybridization using a DNA origami-based molecular scale DNA-coated colloidal assemblies can be rendered light-sensitive by incorporating an azobenzene moiety into the DNA backbone (AzoDNA). Azobenzene switches between cis and trans configurations under UV and blue light respectively. This property enables AzoDNAs to hybridize and dehybridize from each other, facilitating the creation of patterns within the crystal structure upon illumination. However, the single molecule kinetics of this photo switch remain unknown. We determine the lifetimes of individual DNA hybrids by adjusting light intensity, duration of illumination, and the position of azobenzene within the DNA sequence. This is achieved through the design of an origami structure integrated with AzoDNA and utilizing the DNA-PAINT method. Chapter 6: Summary of conducted research and prospects for investigating outstanding questions.