DNA hairpin folding kinetics and mechanisms
Abstract/Contents
- Abstract
- The nucleic acid hairpin structure, composing of a single-stranded loop and a base-paired stem, plays an important role in DNA replication and is a building block of the tertiary structure of large RNA molecules.The biological functions of the hairpin depend on what the state the molecule is in. Thus detailed understanding its folding kinetics and mechanisms facilitates investigations on cellular processes that involve the hairpin structure. DNA hairpin folding has been a subject of intense research, but the current literature does not provide a clear understanding of the folding. This is because that the hairpin folding may involve a large ensemble of intermediate states with vastly different characteristic lifetimes. This unexpected complexity requires advanced techniques that can probe folding in both fast (from nanoseconds to milliseconds) and slow time (more than 1 milliseconds) scales, and previous experiments did not have the advanced techniques to fully probe the folding of the hairpin. The first part of the thesis is a comprehensive study of DNA hairpin folding kinetics. Specifically, we used a multi-faceted and unifying approach that tests each aspect of a general folding model encompassing each of the previously proposed conformational states. Given the complexity in DNA hairpin folding, we have used the following approaches: 1) we extend the temporal range of fluorescence correlation spectroscopy (FCS) by two orders of magnitude to more than 100 mS (for a typical protein). 2) We use both fluorescence quenching and resonance energy transfer to distinguish between proposed structural states; 3) We systematically vary the sequence of the hairpin, both its base pairing and tether regions, to test for the formation of specific intermediate species. 4) We vary ionic conditions to vary the energy landscape and reveal different species and transitions. 5) We follow the behavior of individual molecules for still longer times by surface-tethering molecules and monitoring their folding by con-focal microscopy. Our results reveal a surprisingly simple behavior of a short DNA hairpin of three base pairs. The DNA transitions between a random coil state and a fully folded hairpin, without significant accumulation (< 1%) of misfolded or partially folded intermediate states. It is also discovered that the folding and diffusion processes are coupled, which could be the main factor contributing to the many discrepancies in the literature. Finally, it is revealed that there are three different salt ranges in which the hairpin behaved quite differently. The cations in the solution modulate both the global conformation of the hairpin and the local formation of the stem. In the second part of the thesis, I will present an experimental technique that is called tracking-FRET, which allows extended measurement of molecular conformations by FRET or quenching while tracking the freely diffusing molecular complex in the solution. I will present the basics of the tracking, covering necessary details for us to understand later tracking experiments. Theories to model the tracking system dynamics, compute tracking FRET FCS, and extract the folding dynamics are also presented. Finally, I will present two sets of tracking experiments on the same DNA hairpins we studied in the first part of the thesis. In both experiments, there is overwhelming amount of evidence that we have observed folding dynamics, demonstrating that tracking can be a very powerful tool in learning the folding of macromolecules. However the inconsistency between the tracking and solution FCS results suggests that further development of the tracking apparatus is desired.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Zhang, Ke |
|---|---|
| Associated with | Stanford University, Department of Applied Physics |
| Primary advisor | Mabuchi, Hideo |
| Thesis advisor | Mabuchi, Hideo |
| Thesis advisor | Doniach, S |
| Thesis advisor | Herschlag, Daniel |
| Advisor | Doniach, S |
| Advisor | Herschlag, Daniel |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Michael Ke Zhang. |
|---|---|
| Note | Submitted to the Department of Applied Physics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Ke Zhang
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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