Chapter 3 Sequence Specific Detection of DNA Using Self - Quenched Fluorophore - Polyamide Conjugates

Fluorescent methods for the sequence specific detection of DNA are vital to unraveling the functional, three-dimensional significance of the linear human genome. The ability of minor groove-binding polyamides to specifically target predetermined DNA sequences, without sample denaturation, in biologically relevant condensed states and protein complexes, offers appreciable advantages relative to existing PNAor oligonucleotide-based DNA sensors. Chapter 3 describes the development of fluorophore-polyamide conjugates that exhibit pronounced fluorescent enhancement upon binding their cognate DNA sequences. Chapter 3A discusses the synthesis and physical characterization of TMRhairpin polyamide conjugates. The fluorescent properties of these compounds in the absence of DNA and in the presence of both match and mismatch DNA duplexes were evaluated. The sequence specificity of fluorescent enhancement was validated by correlation with observed association constants determined by quantitative DNase I footprinting. The use of TMR-hairpin conjugates to recognize non-Watson-Crick base pairs was also demonstrated. Chapter 3B suggests applications of fluorophore-polyamide conjugates in genomic analysis. Centromere staining in the context of cytogenetic chromosome spreads, as well as in live cells, is discussed and the detection of other repeat sequences is proposed. Preliminary experiments addressing the suitability of polyamides in physical methods of DNA mapping are summarized.