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Abstract
RNA interference (RNAi) molecules have become a powerful drug development platform and are critical tools in molecular biology. They harness the endogenous RNAi defense mechanism to destroy mRNA or repress its translation. The interference is sequence specific and highly selective. For this reason, small interfering RNA (siRNA) have become indispensable tools for elucidating signaling pathways and studying disease states. siRNA are also being used as potential therapeutics and occupy an increasingly prominent place in preclinical drug trials. For example, more than 50 oligonucleotides have been submitted for clinical trials, and 11 of these are siRNAs. Potential uses for siRNA include treatment of respiratory syncytial virus, Hepatitis B, human immunodeficiency virus (HIV) as well as Huntington's Disease and cancer. The increased use of siRNA has highlighted deficits in our ability to accurately analyze and quantify these molecules. While traditional hybridization assays provide excellent sensitivity, they often do not distinguish between the parent and metabolites. There is a specific need for sensitive methods that allow for the quantitation of siRNA and its chain-shortened metabolites for pharmacokinetic and pharmacodynamic analysis as well as metabolism studies that assist in the rational design of modifications of siRNA therapeutics. Chapter one is an introduction which provides the layout for the dissertation. Chapter 2 provides a review of the literature for chromatographic methods for oligonucleotides. This review highlights the need for more sensitive methods for bioanalysis including simplified sample preparation procedures. Chapter 3 covers the development of a novel ion-exchange method with a one step sample preparation procedure. It also covers the analysis of the uptake of siRNA in cell culture and the identification of siRNA metabolites by mass spectrometry. Low-level detection of metabolites requires specific and sensitive methods for liquid chromatography with mass spectrometry detection. Chapter 4 covers the evaluation of alkylamines and hexafluoroisopropanol for their effect on electrospray desorption efficiency on siRNA and unmodified DNA. A model for the prediction of optimized mobile phase composition is proposed based on the chemical properties of the oligonucleotide.