I present two applications of fluorescence spectroscopy in biophysics. The first is an instrument, the anti-Brownian electrokinetic (ABEL) trap, which is capable of trapping individual small molecules in aqueous solution at room temperature. The second is an investigation of the bending mechanics of double-stranded DNA using a novel DNA structure called a "molecular vise". Both projects take advantage of the sensitivity and specificity of fluorescence spectroscopy, and both benefit from the interplay of experimental work with theoretical and computational modeling. The ABEL trap uses fluorescence microscopy to track a freely diffusing particle, and applies real-time electrokinetic feedback forces to oppose observed motion. Small molecules are difficult to trap because they diffuse quickly and because their fluorescence emission is typically weak. I describe the experimental and algorithmic approaches that enabled small-molecule fluorophores to be trapped at room temperature. I additionally derive and discuss the theory of the molecules' behavior in the trap; this mathematical work informed the design of the trapping algorithm and additionally enabled trapped molecules to be distinguished on the basis of their diffusion coefficient and electrokinetic mobility. Molecular vises are DNA hairpins that use the free energy of hybridization to exert a compressive force on a sub-persistence length segment of double-stranded DNA. In response to the applied force, this "target strand" may either remain straight or bend, depending on its flexibility and length. Experimentally, the conformation can be monitored via Förster resonance energy transfer (FRET) between appended fluorophores. The experimental results quantitatively matched the predictions of the classic wormlike chain (WLC) model of DNA elasticity at low-to-moderate salt concentrations. Higher ionic strength induced an apparent softening of the DNA which was best accounted for by a high-curvature "kinked" state. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA and provides a platform for studying the effects of chemical modifications and small-molecule or protein binding on these properties.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/11129111 |
| Date | January 2013 |
| Creators | Fields, Alexander Preston |
| Contributors | Cohen, Adam Ezra |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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