Dissertation Defence: The Development of DNA-based Tools to Investigate Biomolecular Interactions Under Force
May 29 at 9:00 am - 1:00 pm
Adam Yasunaga, supervised by Dr. Isaac Li, will defend their dissertation titled “The Development of DNA-based Tools to Investigate Biomolecular Interactions Under Force” in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology.
An abstract for Adam Yasunaga’s dissertation is included below.
Examinations are open to all members of the campus community as well as the general public. Registration is not required for in-person exams.
Abstract
Mechanical forces at the molecular scale play critical roles in regulating cellular behaviour, yet methods for measuring and applying these forces remain limited. In particular, highly dynamic and fast interactions are a challenge to study. This thesis addresses these challenges by developing new methodologies for the detection, quantification, and application of molecular forces with high precision and versatility.
The research begins with a comprehensive review of molecular force sensors, classifying current designs based on their mechanical properties and fluorescent readout mechanisms. Building on this foundation, an adhesion footprint assay that utilizes irreversible molecular force sensors was developed to quantify the forces experienced by P-selectin: PSGL-1 interactions during rolling adhesion. Complementary statistical modelling of rolling adhesion behaviour relates instantaneous rolling velocity distributions to underlying molecular force distributions, providing new insights into the mechanical behaviour of the rapid adhesion events involved in rolling adhesion.
In the latter part of the thesis, the focus shifts toward tool development for force application. The Flow Force Assay was introduced as a high-throughput single molecule force spectroscopy technique based on hydrodynamic flow-stretching of DNA. By using flow-stretched DNA as a force generator within microfluidic channels, controlled forces were applied to molecular interactions with nanometer-scale spatial resolution. To enable quantitative force measurements, an empirical model was developed to calibrate the relationship between DNA extension, wall shear stress and piconewton scale forces. Calibration was validated experimentally across multiple DNA constructs, achieving reliable force quantification up to 20 pN.
Together, the methods developed in this thesis expand the experimental capabilities for studying mechanical forces at the molecular scale. The combination of irreversible molecular force sensing, flow-based force application, and precise calibration enables the interrogation of molecular adhesion and mechanotransduction processes that were previously inaccessible. Future work will focus on extending the validated force range of the Flow Force Assay and applying these methods to more complex biomolecular interactions. This work contributes to advancing molecular force spectroscopy and provides new tools for understanding the mechanical component of cell biology.
