Force spectroscopy of nucleic acid folding in the single-molecule limit

  • Author / Creator
    Foster, Daniel A N
  • Folding of biomolecules is an important problem in structural biology. The physical folding can be projected as a diffusive search over an energy landscape whose dimensions scale by all the internal degrees of freedom which a biomolecule possesses. To explore this idea, folding is studied from the perspective of its biological relevance in RNA, and then the specifics of folding as a physical process in nucleic acids with a particular focus on DNA as a model system. For studying the relationship between folding and function, I focus on two classes of RNA regulatory molecules whose structural dynamics are integral to their function. The first, a riboswitch, is a sequence within a messenger RNA (mRNA) that regulates gene expression. It does this by ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. The second class of molecules is the pseudoknot, a structure also found in mRNA that promotes shifting of the reading frame of the ribosome. These RNA molecules are studied by single-molecule force spectroscopy using optical tweezers, to repeatedly unfold and refold them in order to explore the conformations they form. The study of the add riboswitch reveals a thermodynamically-controlled regulatory mechanism and a rare misfolding pathway. The study of a panel of programmed −1 frameshifting pseudoknots reveals that their frameshifting efficiency does not correlate with mechanical resistance to unfolding but instead with the tendency towards the formation of alternate, incompletely folded structures. To explore the physical process of folding in detail, and to make quantitative tests of diffusive theory, we use DNA hairpins that have been previously well characterised. The first study is to determine the transition times between two states in a folding/unfolding system from energy landscape analysis, then by direct measurement. These trajectories dominate the dynamics of the folding reaction, encapsulating critical information about how structure forms. New methods proposed for measuring the position-dependence of the diffusion coefficient are tested using the round-trip time with equilibrium data, both experimental and simulated, and by using the average fall time from non-equilibrium force jump experiments. The results deviate from expectation, as well as producing different results when started from the folded state or from the unfolded state. Finally, a novel method proposed for the landscape reconstruction from discontinuous force-jump experiments is explored with both the assumption of a constant diffusion and one that is position dependent. For both of these methods it is found that they do not replicate the results from other techniques. For all these new methods, their disagreement seems most likely because they are sensitive to or do not consider the compliance and dynamics that beads and handles introduce into the force probe.

  • Subjects / Keywords
  • Graduation date
    Fall 2015
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Klobukowski, Mariusz (chemistry)
    • Morsink, Sharon (physics)
    • Li, Hongbin (chemistry - university of british columbia)
    • Tuszynski, Jack (physics)
    • Freeman, Mark (physics)