The role of secondary structure in DNA recognition as applied to pathogen detection

The dimerization of complementary strands of DNA is one of the premier examples of evolution's ability to harness the power of molecular recognition. The seemingly simple nature of these dimerization events belies the level of complexity that is actually involved, and developing a better understanding of these intricacies will have far-reaching effects on humanity's ability to develop highly sensitive and selective DNA recognition probes.; The most efficient way to harness the recognition capabilities of DNA is to incorporate it into a high density array. However, immobilizing DNA intrinsically alters the nature of the hybridization process.{09}With the aim of developing our understanding of how surface immobilized DNA interacts with its solution-phase target, we have developed two optical methods for the label-free detection of oligonucleotides. The first method, reflective interferometry, can incorporate linear DNA probes and accurately detect viral DNA. We have also developed a technique where fluorescently labeled DNA hairpins were immobilized onto a metal surface allowing for a systematic study of the function of shape in DNA recognition.; In an effort to increase the ability to accurately serotype a variety of pathogens, we have developed a novel method for the discovery of naturally occurring hairpins. This method, based on the subjection of large segments of species specific DNA to a computer folding algorithm, has been demonstrated to be a vast improvement over the traditional method of hairpin generation because of its elimination of the need to supplement a target DNA sequence with non-specific oligonucleotides. Additionally, many of these naturally occurring hairpins are predicted to have a very high level of secondary structure, allowing us the ability to elucidate the role structure plays in either promoting or preventing DNA dimerization. To date, our new method has allowed for the discovery of hairpins from Bacillus anthracis and Staphylococcus aureus that are both highly functional and highly specific. These hairpins have been studied both in solution using UV-Vis and fluorescence spectroscopy, and immobilized on a solid surface, using an Au film as both a support and a quenching agent for the distal fluorophore.