Fluorescence Probing of DNA Structures in Biology and Nanotechnology

Doctoral thesis, 2008

Much has been learnt about DNA since its structure was discovered about half a century ago. Scientists everlasting urge to look beyond and deeper into the intricate machinery of life, has continuously pushed technology forward. This Thesis focuses on the development of two new DNA probes, tC and tCO, that both show promises of opening up new possibilities for fluorescence studies of DNA and DNA containing systems. They are fluorescent DNA base analogs, a group of probes with the appealing ability to mimic the natural bases which allows for virtually non‐perturbing substitution into DNA. The cytosine analogs tC and tCO with their minimal effect on the DNA native structure and unique fluorescence properties, offer significant improvements on previously reported base analogs and are especially suited for techniques such as fluorescence resonance energy transfer (FRET), fluorescence anisotropy and fluorescence detected DNA melting (tCO). This Thesis presents a careful characterization of their effect on DNA structure and stability, their photophysical properties, both as monomers and when incorporated into DNA, in addition to demonstrating some examples of their potential as fluorescent DNA probes. The Thesis also explores a new approach to self‐assembled DNA nanoarrays. The approach is intended to allow for the construction of a non‐repetitive and addressable nanoarray with unprecedented information density. As the fundamental pseudohexagonal unit‐cell of this array is significantly smaller than previously published ones, finding ways to characterize this structure has been one of many challenges and a primary focus in this Thesis. The successful and high yield of formation of the fundamental unit was verified using a combination of electrophoresis and spectroscopic techniques. Time‐resolved FRET was shown to be a useful alternative to the microscopy techniques commonly used for larger structures. It was used for retrieving structural information on the pseudo‐hexagon in addition to revealing that this motif has a large conformational flexibility. Details on the formation, melting and stability of the pseudohexagon were obtained by using tCO as a fluorescent probe for DNA melting. This led to a clearer understanding of the thermodynamics governing the formation of the pseudohexagonal structure which will aid in the design and formation of future DNA nanostructures and arrays.