New Biophysical Assays to Study Biomacromolecular Folding, Assembly, and Function

• Main Author: Hennecker, Christopher D
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2024
• Subjects: Biology; Biophysics; Deoxyribonucleic acid; DNA; Genes; Genetics; Information storage; Kinetics; Mathematics; Medical imaging; NMR; Nuclear magnetic resonance; Ordinary differential equations; Physical properties; Polymorphism; Ribonucleic acid; RNA; Sugar; Thermodynamics
• ISBN: 9798342117029

Biomacromolecules are fundamental in virtually all aspects of biology. Developing a deep understanding about how these complex molecular systems behave is crucial for understanding their biological function and exploiting their physical properties for new technologies. For example, the self-assembly of these biomacromolecules can produce interesting materials which exhibit emergent physical properties such as self-healing and stimuli responsiveness. Furthermore, both enzymes and non-canonical DNA secondary structures are popular targets for small molecule therapeutics. However, in order to develop these molecules, scientists need to know how stable these structures are, how strongly they interact with their targets, and how to improve their desired properties. However, due to their highly complex nature, there are many examples of when conventional experimental techniques fail. Thus, new methods which enable quantitative characterization of these more complex systems are highly desirable.This thesis explores novel biophysical analyses that combine common laboratory equipment with modern day computational power and mathematical modelling to address the need for rapid and cost-effective biomacromolecular characterization. Chapter 2 details a global-fitting analysis for non-equilibrium thermal denaturation experiments and its application to the folding dynamics of guanine quadruplexes (G4s), four stranded non-canonical nucleic acid structures formed from guanine rich sequences that are implicated in wide variety of cancers. We demonstrate that these sequences can fold into several different structures via parallel pathways which significantly increases their folding rate, potentially influencing their biological function.Chapter 3 proposed the concept of G4 containing regions (G4CRs). We then developed a bioinformatic algorithm which characterizes these G4CRs based on their length and total number of G4 structures. This algorithm was applied to human promoter sequences where we found G4CRs that were up to several hundred nucleotides long and had the potential to form thousands of G4 structures. These polymorphic G4CRs were clustered directly adjacent to the transcription start site, suggesting that polymorphism has a functional role in biology.In Chapter 4, we developed an experimental approach based off cyclic heating and cooling ramps which can measure thermodynamic information on slowly assembling supramolecular structures, information which was previously unobtainable with any other method. We used this technique to study the co-assembly of poly-adenosine strands and cyanuric acid (CA) into long supramolecular fibres. We discovered that roughly one third of CA binding sites were unoccupied, which has implications for the use of this system as a drug delivery vehicle.Finally, Chapter 5 describes how to measure the binding kinetics of covalent inhibitors using isothermal titration calorimetry (ITC). Our new method allowed for faster and more robust characterization of these inhibitors when compared to conventional methods and removes the need for modified or spectroscopically active substrates as ITC is able to directly measure the rate of enzymatic catalysis. Together, these approaches represent powerful new additions to researchers' toolkit for rigorous characterizations of biomacromolecular folding, assembly, and function