Student Theses and Dissertations

Date of Award

2000

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Libchaber Laboratory

Abstract

This thesis is centered on the development of the molecular beacon, as a new DNA probe for DNA genotyping, D N A computation and biophysical studies of DNA conformations. Molecular beacons are single-stranded DNA molecules that form a stem-and-loop structure. A fluorophore and a quencher are grafted at their two ends to report their conformations: when the molecular beacon is closed, fluorophore and quencher are held in close proximity and the fluorescence is quenched; when the molecular beacon is open, fluorophore and quencher are far apart, and the fluorescence is restored. Molecular beacons are ideal DNA probes coupling conformational switch with fluorescence signal turning-ON. We use molecular beacons to study the molecular recognition of single-stranded DNA (ssDNA) oligonucleotide. We present a thermodynamic diagram to show that structural constraints make the molecular beacon highly sensitive to the presence of mismatches in its target. We introduce a sequence sensitivity parameter to quantitatively compare different DNA probes, and propose an algorithm to optimally tune the probe's structure for enhanced sequence discrimination. Logic gates (OR and AND gates) using molecular beacons are designed to carry most elementary molecular computations. The conformational changes associated with such computations can be used to concatenate many chemical reactions, and carry out complex molecular computations. Molecular beacons are also ideal probes to study DNA secondary structures and their fluctuations. We develop the fluorescence correlation spectroscopy (FCS) technique to monitor the dynamics of relaxation of DNA conformational fluctuations. We first measure the opening and closing timescales of DNA hairpin-loops. Activation barriers for opening and closing for different loop lengths and sequences are analyzed to better account for the stability of DNA secondary structures. A sequence dependent rigidity of ssDNA has been discovered, and analyzed in terms of base stacking. We then use F C S to study the dynamics of double-stranded DNA (dsDNA) breathing modes with synthetic DNA constructs. The analysis of the base pairing fluctuation dynamics, monitored by fluorescence, unravels lifetimes of breathing modes ranging from 1/us to 1ms. Long-range distortions of the d s DNA have been unraveled for purine-rich sequences, of relevance to the specificity of transcription initiation in prokaryotes.

Comments

A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy At The Rockefeller University

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