Design of ligands for sequence-specific recognition of the minor-major grooves of DNA

Citation

Szewczyk, Jason W. (1999) Design of ligands for sequence-specific recognition of the minor-major grooves of DNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/zt1g-kq41. https://resolver.caltech.edu/CaltechETD:etd-04282006-112749

Abstract

The two most powerful chemical approaches to date for the sequence-specific recognition of double helical DNA are the pyrrole-imidazole polyamides in the minor groove and oligonucleotide-directed triple-helix formation in the major groove. Described here are the design and synthetic methods to combine the two models for DNA recognition in a single motif. A hairpin polyamide connected to a pyrimidine-oligonucleotide via a simple aliphatic linker was shown to simultaneously recognize the major and minor grooves of DNA at subnanomolar concentrations (Chapter 2). Development of versatile solid phase methodology expanded the protocols for the synthesis of pyrrole-imidazole polyamides to include polyamide-oligonucleotide conjugates. Utilizing the pyrrole-imidazole polyamide moiety as a sequence-specific dimerization domain afforded a class of cooperative polyamide oligonucleotides which bound a 27 bp target with a 2.7 kcal mol(-1) increase in binding energy relative to the unlinked subunits (Chapter 3). Introduction of a new linker design created an extended polyamide-oligonucleotide motif to specifically target 31 contiguous base pairs of DNA at subnanomolar concentrations (Chapter 4). Polyamide-oligonucleotides combine the two binding models to create a paradigm for simultaneous recognition of the major-minor grooves of DNA with synthetic ligands.

For the pyrrole-imidazole polyamide model, sequence specificity depends on side-by-side aromatic amino acid pairings. An Im/Py pair distinguishes G • C from C • G and both of these from A,T base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish A•T from T•A. To break this degeneracy a new aromatic amino acid, 3-hydroxypyrrole (Hp), was synthesized to test for pairings which discriminate A • T from T • A. Replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of a third aromatic amino acid, hydroxypyrrole-imidazolepyrrole polyamides form four ring pairings (Im/Py, Py/Im, Hp/Py, Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA (Chapter 5).

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