Sequence-specific recognition of DNA by pyrrole-imidazole hairpin polyamides

Citation

Parks, Michelle E. (1996) Sequence-specific recognition of DNA by pyrrole-imidazole hairpin polyamides. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/4cd1-s277. https://resolver.caltech.edu/CaltechETD:etd-04282006-114140

Abstract

Polyamides composed of pyrrole (Py) and imidazole (Im) amino acids bind as antiparallel, side-by side dimers in the minor groove of DNA to sequences containing both (A,T) and (G,C) base pairs. Initial polyamides based on the 2:1 model have demonstrated new, predictable specificities but only modest affinities. Chapter 2 describes the thermodynamic characterization of a series of covalently linked polyamides, revealing an increase in affinity (>300-fold) and specificity of the hairpin compared to the unlinked dimers. Chapter 3 reports the characterization of the corresponding cyclic polyamide, which results in a 40-fold increase in affinity, although specificity decreases.

Chapter 4 explores the generality of the 2:1 polyamide:DNA model by systematically varying the imidazole content and position within a series of eight three-ring polyamides. Of ten homodimeric and heterodimeric combinations of polyamides characterized, four bind as expected, indicating that the 2:1 model, while not completely general, is a valuable predictive tool. This work adds three new sequences to the targetable repertoire for polyamides, 5'-WGGWW-3', 5'-WGGCW-3', and 5'-WCGCW3'. Chapter 5 focuses on a series of linked hairpin analogs of the heterodimer AcImImPyDp/distamycin that recognize 5'-WGGWW-3' sequences. This study provides the first example of sequence-specific recognition of contiguous G•C base pairs using the side-by-side polyamide:DNA model. In addition, footprinting experiments reveal that linker position does not significantly affect the affinity or specificity of hairpin polyamides. Chapter 6 describes the optimization of polyamide N- and C-terminal groups. Thermodynamic characterizations of a series of ten polyamides show that an unacetylated N-terminus and a C-terminal [beta]-alanine spacer are optimal for hairpin polyamides, providing important design guidelines.

Chapter 7 examines the simultaneous binding of an oligonucleotide in the major groove of DNA and a polyamide in the minor groove. Quantitative footprinting experiments indicate that the stability of a triple helix is not affected by simultaneous recognition in the minor groove, suggesting that oligonucleotide-polyamide conjugates could be designed for sequence-specific DNA recognition. Chapter 8 describes experiments designed to determine the DNA-binding orientation of a portion of a high mobility group protein. Although the synthetic peptides bind A,T-rich DNA, no specific cleavage was observed, precluding determination of binding orientation.

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