Design of DNA-binding polyamides for regulation of gene expression.

摘要

Polyamides containing N-methylimidazole and N-methylpyrrole amino acids are synthetic ligands that bind DNA by forming side-by-side complexes in the minor groove. Sequence-specificity depends on side-by-side aromatic amino acid pairings: imidazole (Im) opposite pyrrole (Py) targets a G.C base pair, Py/Im targets C.G and Py/Py targets T.A and A.T. This thesis describes work centered on the design of polyamide motifs that increase the binding site size limit, binding affinity and sequence repertoire of polyamides. In addition, polyamides were designed to bind within the regulatory regions of selected genes, which allowed investigations of polyamides as regulators of gene expression.;Chapter Four describes eight-ring hairpin polyamides that bind respective six base pair target sequences with subnanomolar affinity and discriminate match from single-base pair mismatch sites. These results showed that polyamides can bind predetermined DNA sequences with affinity and specificity comparable to DNA-binding gene-regulatory proteins.;In Chapters Five and Six, studies of hairpin polyamides that incorporate beta-alanine (beta) residues to correctly position internal imidazoles showed that the choice of a Py/Py, Py/beta or beta/beta pair depends on the sequence composition of the target site. These results expanded the sequence repertoire of polyamides and contributed to polyamide design guidelines that predict, for a given target sequence, when and where beta-alanine residues are needed.;Chapter Eleven describes polyamides that bind DNA sequences proximal to transcription factor binding sites within the promoter regions of the 5S rRNA gene, the HIV-1 genome and the HER2/neu gene. The results of biological assays using these polyamides demonstrated that polyamides are cell-permeable, can interfere with transcription factor-DNA interactions and can inhibit the transcription of specific genes and viral replication within living cells.;In Chapter Two, beta-alanine was found to optimally link three-ring polyamide subunits in an extended conformation, allowing recognition of 9 and 13 base pair sequences. This result extended the binding site size limit of polyamides and identified a useful polyamide building block. Chapter Three describes the ability of C-terminal amino acids to modulate the DNA-binding affinity and specificity of polyamide dimers. Chapters Seven, Eight, Nine and Ten describe additional polyamide motifs for recognition of 9--16 base pair sequences.