Single molecule methods for DNA-protein interaction studies
Doctoral thesis, 2025
In the volume of a typical cell, even a single molecule significantly affects the concentration. Therefore, at typical affinities, only a few proteins are required to saturate binding to, for example, DNA. Compartmentalization further lowers this threshold, underscoring the need for sensitivity and molecular recognition and highlighting the importance of precision in biological research.
Given the small number of molecules required to reach functional concentrations in a cell, it becomes clear that studying molecular interactions at the single-molecule level can provide important insights. However, studies of biochemical reactions, such as DNA-protein interactions, are typically done with bulk methods. This approach is robust, but the result is based on a time and population average of all the molecules present in the reaction. If multiple populations co-exist, the average can be misleading. By isolating molecules so that they can be studied one-by-one, as in single-molecule methods, it is possible to resolve separate populations that otherwise would have been hidden.
The original work presented in this Thesis explores [FW6] the application of single-molecule methods for studying DNA-protein interactions. The work is focused on the use of quantitative fluorescence microscopy and involves the use of nanofluidics to study the DNA-repair protein complexes MRN (human) and MRX (yeast), and the role of their individual components in DNA end-joining. The potential of nanofluidics was further explored by establishing the influence of divalent metal ions and ATP on the binding of the fluorescent dye YOYO-1 to DNA and the impact this has on studies of active protein processes on DNA. The Thesis also encompasses work involving DNA-protein complexes immobilized on functionalized glass slides. It presents a novel quantitative method where colocalization with a fluorescent dCas9 is used to identify and size viral vectors isolated from cells. Furthermore, the same principle is used in a study showing that the oncogenic protein MYC is involved in alternating the activity of Topoisomerase 2A by co-condensation. Taken together, the work presented in Thesis provides valuable insights in how to isolate and immobilize DNA-protein complexes and combine this with quantitative fluorescence microscopy to extract meaningful biological insights from single-molecule data.
Given the small number of molecules required to reach functional concentrations in a cell, it becomes clear that studying molecular interactions at the single-molecule level can provide important insights. However, studies of biochemical reactions, such as DNA-protein interactions, are typically done with bulk methods. This approach is robust, but the result is based on a time and population average of all the molecules present in the reaction. If multiple populations co-exist, the average can be misleading. By isolating molecules so that they can be studied one-by-one, as in single-molecule methods, it is possible to resolve separate populations that otherwise would have been hidden.
The original work presented in this Thesis explores [FW6] the application of single-molecule methods for studying DNA-protein interactions. The work is focused on the use of quantitative fluorescence microscopy and involves the use of nanofluidics to study the DNA-repair protein complexes MRN (human) and MRX (yeast), and the role of their individual components in DNA end-joining. The potential of nanofluidics was further explored by establishing the influence of divalent metal ions and ATP on the binding of the fluorescent dye YOYO-1 to DNA and the impact this has on studies of active protein processes on DNA. The Thesis also encompasses work involving DNA-protein complexes immobilized on functionalized glass slides. It presents a novel quantitative method where colocalization with a fluorescent dCas9 is used to identify and size viral vectors isolated from cells. Furthermore, the same principle is used in a study showing that the oncogenic protein MYC is involved in alternating the activity of Topoisomerase 2A by co-condensation. Taken together, the work presented in Thesis provides valuable insights in how to isolate and immobilize DNA-protein complexes and combine this with quantitative fluorescence microscopy to extract meaningful biological insights from single-molecule data.
nanofluidics
biomolecular interactions
DNA repair
DNA
fluorescence microscopy
single molecule
10:an, Forskarhus 1, Kemihuset
Opponent: Prof. Jonas Tegenfeldt, Department of Physiscs, Lund, Sweden
Author
Carl Ivar Möller
Chalmers, Life Sciences, Chemical Biology
Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex
Biochemical and Biophysical Research Communications,;Vol. 695(2024)
Journal article
Carl Möller, Dennis Winters, Radhika Nambannor Kunnath, Sriram KK, Fredrik Westerlund Effects of Mg2+ and ATP on YOYO-1 labelling of genomic DNA in single molecule nanofluidic experiments
Carl Möller, Luis Leal-Garza, Emanuele Celauro, Roberto Nitsch, Fredrik Westerlund Cas9-leveraged single-molecule characterization of sparse plasmid vectors in heterogenous DNA samples
Donald Cameron, Kathryn Jackson, Alessia Loffreda, Carl Möller, Matteo Mazzocca, Evgeniya Pavlova, Bea Jagodic, Fredrik Westerlund, Davide Mazza, Laura Baranello MYC modulates TOP2A diffusion to promote substrate detection and activity
Single-molecule microscopy: studying the straws in the haystack
Imagine trying to understand how a machine works by watching thousands of its parts moving at once. You might get an average idea, but not be able to tell which parts are doing what, or if some are malfunctioning. This is a challenge often met when studying life on the molecular scale. How proteins interact with DNA is key for understanding e.g. DNA repair, a process vital for genome stability. However, many methods average the behavior of millions of molecules, missing rare events and subtle differences. The included work shows howstretching DNA in nanochannels or immobilizing it on glass slides enables the use offluorescence microscopy to visualize single DNA molecules, revealing what is hidden in the crowd.
A study on DNA-repair protein complexes from humans and yeast highlights how the individual components contribute to holding broken DNA in place, an important step in DNA repair. A second study, on the impact of multivalent ions and ATP on the DNA-specific fluorescent dye YOYO-1, provides guidance for future single-molecule work. The Thesis further introduces a novel method using Cas9 to detect and size single DNA molecules in mixed samples. Finally, the work explores how MYC, a cancer-related protein, influences the formation of DNA-protein-condensates, helping cells manage stress from high transcription activity.
In summary, this thesis contributes to the development of methods that focus on the individual straws in the haystack, potentially even finding the needle.
Imagine trying to understand how a machine works by watching thousands of its parts moving at once. You might get an average idea, but not be able to tell which parts are doing what, or if some are malfunctioning. This is a challenge often met when studying life on the molecular scale. How proteins interact with DNA is key for understanding e.g. DNA repair, a process vital for genome stability. However, many methods average the behavior of millions of molecules, missing rare events and subtle differences. The included work shows howstretching DNA in nanochannels or immobilizing it on glass slides enables the use offluorescence microscopy to visualize single DNA molecules, revealing what is hidden in the crowd.
A study on DNA-repair protein complexes from humans and yeast highlights how the individual components contribute to holding broken DNA in place, an important step in DNA repair. A second study, on the impact of multivalent ions and ATP on the DNA-specific fluorescent dye YOYO-1, provides guidance for future single-molecule work. The Thesis further introduces a novel method using Cas9 to detect and size single DNA molecules in mixed samples. Finally, the work explores how MYC, a cancer-related protein, influences the formation of DNA-protein-condensates, helping cells manage stress from high transcription activity.
In summary, this thesis contributes to the development of methods that focus on the individual straws in the haystack, potentially even finding the needle.
Next Generation Nanofluidic Devices for Single Molecule Analysis of DNA Repair Dynamics
European Commission (EC) (EC/H2020/866238), 2020-04-01 -- 2025-03-31.
Real-Time Visualization of DNA Repair - One Molecule at a Time
Swedish Research Council (VR) (2020-03400), 2021-01-01 -- 2024-12-31.
Subject Categories (SSIF 2025)
Molecular Biology
Nanotechnology for/in Life Science and Medicine
Biophysics
Areas of Advance
Health Engineering
ISBN
978-91-8103-243-7
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5701
Publisher
Chalmers
10:an, Forskarhus 1, Kemihuset
Opponent: Prof. Jonas Tegenfeldt, Department of Physiscs, Lund, Sweden