Using nucleic acid chemistry to probe telomeric DNA function and structure

Telomeres are the ends of linear chromosomes in eurkaryotic organisms and represent a critical mechanism that regulates cellular growth and death. This dissertation relates efforts to study the structure and function of telomeres and telomeric DNA through the application of nucleic acid chemistry.; In the first part, we describe a method to elongate repetitive telomeric DNA sequences through the use of a rolling-circle reaction in which small circular single-stranded DNA oligonucleotides ('nanocircles') are used as templates by polymerases. Using an assay based on fluorescent nucleotide incorporation, we found that circles encoding telomeric sequence could be used in vitro to elongate real telomeres extracted from human cells using several polymerases. We also investigated the telomere length of primary cell lines following transfection with DNA nanocircles encoding telomeric sequence. Measurements by Quantitative Fluorescence in situ Hybridization and Southern Blot experiments showed no significant differences between the telomere length of nanocircle-treated cells relative to controls. Future experiments studying the replicative lifespan of cells after nanocircle treatment and a more detailed investigation of the cellular fate of transfected nanocircles should provide more information on possible elongation by nanocircles.; In the second part, we describe efforts to probe the specific structures formed by G-rich telomeric oligonucleotides. We examined a series of human telomeric oligonucleotides of different length by UV spectroscopy and circular dichroism. We observed a higher ratio of 'parallel' to 'antiparallel' folding by circular dichroism with increasing sequence length, and confirmed the tendency of these oligonucleotides to form preferentially 'parallel' folds in K + versus Na+ solutions. The folds formed by longer telomeric sequences also appeared to be more stable in K+ than in Na+ for oligonucleotides of the same length. We hypothesize that this unexpected stability is due to possible stacking interactions between individual quadruplex subunits. We also prepared perylene- and pyrene-labeled telomeric oligonucleotides in the hope of observing interactions between these fluorophores when specific hypothesized structures were formed. We observed energy transfer between pyrene and perylene labels in telomeric oligonucleotides that were predicted to allow this interaction, establishing this method as a possibly useful tool for the determination of telomeric DNA structure.