Investigating the precision of dynamic DNA origami platforms with multi-color single-molecule FRET methods and using them to benchmark deep-neural networks analysis approaches

Asadiatouei, Pooyeh (2024): Investigating the precision of dynamic DNA origami platforms with multi-color single-molecule FRET methods and using them to benchmark deep-neural networks analysis approaches. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Many fundamental biological processes and interactions can be successfully explored by applying Förster resonance energy transfer (FRET) techniques which have become crucial tools in molecular biology and biophysics. By strategically incorporating suitable dye pairs into different positions of biomolecules, we can resolve both inter- and intra-molecular distances, as well as dynamic interactions with sub-nanometer resolution. Such level of detail is essential for understanding the molecular machinery of life, as it allows us to observe interactions and conformational changes that are often invisible when using traditional methods. FRET is particularly powerful due to its extreme sensitivity to the distance between the centers of molecules since it is related to the 6th power of the distance, so it is one of the most effective tools for detecting small changes. Unlike conventional ensemble measurements, which average out molecular behaviors across an ensemble, single-molecule FRET (smFRET) enables the detection of subtle variations such as conformational and functional heterogeneities within a sample. These underlying variations provide critical insights into the complex behaviors of biomolecular systems. The ability to observe such differences with the appropriate statistics, can uncover hidden states, transient interactions and rare events that play key roles in biological functions. Traditionally, FRET experiments were focused on two-color systems, where a donor and acceptor dye pair report on a single distance. However, recent advancements have pushed the boundaries of FRET applications by introducing three-color FRET assays. Incorporating three labels into the system allows for the simultaneous measurement of multiple distances, creating a more comprehensive three-dimensional vision of the molecular interactions and structures. This multi-color approach not only helps track individual distances between each dye pair but also reveals correlations between them, offering a richer understanding of the molecular dynamics under study. Although three-color FRET assays can complicate both the experimental design and data analysis, these challenges have been addressed. Thanks to significant advancements in instrumentation and the development of sophisticated analysis software, the once intimidating task of analyzing three-color FRET data has become more manageable. The technological innovations in detection sensitivity, data processing and automation have transformed what was once a time-consuming and user-prone process into a streamlined, reliable workflow. As a result, researchers can now perform complex three-color FRET experiments with reduced analysis times, removing barriers that previously might have discouraged the widespread adoption of such techniques. The combination of solution-based and surface-based smFRET assays offers a versatile approach for studying biomolecular systems. Solution-based assays are ideal for capturing the fast dynamic behavior of molecules in their native states and not bound to the surface, while surface-immobilization assays allow for long-term observations of molecular kinetics by fixing molecules on a chamber surface area. By employing both techniques, we can gain a comprehensive understanding of the sample, taking advantage of the different time scales and environmental conditions each method provides. This dual approach enables the examination of individual biomolecules behavior in ways that might be impossible using just one of the techniques. A particularly exciting development in the field of smFRET is its combination with DNA origami nanotechnology. DNA origami structures allow for the precise design and construction of nanoscale samples, and have revolutionized many areas of single-molecule biophysics. When complemented with smFRET, DNA origami provides a versatile platform for positioning fluorophores with sub-nanometer precision, enabling extreme control over the spatial arrangement of dyes. This level of control facilitates the study of a wide range of fundamental questions, from unraveling the structural features and distances, and molecular functions to probing the single fluorophores behavior in dynamic molecular changes and interactions. In this thesis, both two- and three-color smFRET assays were employed to benchmark a powerful and versatile analysis tool based on deep learning. The primary goal was to significantly shorten the data analysis time and eliminate user bias during the analysis process, which is a common issue in manual data interpretation. The development of this tool has reduced the time required for analysis from weeks to minutes, revolutionizing how data is processed and visualized, and open a whole new world of possible experiments. Moreover, the experiments conducted on various L-shaped DNA origami nanostructures, which were integral to the development of these smFRET assays, uncovered a number of interesting characteristics related to the behavior of fluorophores and labeling strategies. These findings have prompted us to further investigate the precise control and manipulation of fluorophore positioning and transient binding kinetics of single-stranded DNA in the context of the DNA origami structure. A comprehensive set of experiments was executed to explore how accurately and consistently one can arrange and control the transient binding kinetics of DNA single strands. These studies revealed a number of factors that can interfere with the behavior and kinetics of the system, including the type of fluorophores used and the specific labeling or binding positions. By understanding and characterizing such factors, we can now predict and control the performance of FRET-based assays more reliably.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 28. Oktober 2024
• 1. Bericht­erstatter:in: Lamb, Don C.