The Molecular Recognition of DNA by Rhodium(III)-Zinc Finger Peptide Chimeras

Citation

Lin, Susanne Chosein (1997) The Molecular Recognition of DNA by Rhodium(III)-Zinc Finger Peptide Chimeras. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/mss5-wf97. https://resolver.caltech.edu/CaltechTHESIS:07182025-180545892

Abstract

Covalent chimeras of zinc finger peptide domains with phenanthrenequinone diimine (phi) complexes of rhodium (III) have been designed, synthesized and their DNA recognition characteristics examined. The rhodium complex binds in the major groove of DNA by intercalation and allows the attached peptide to interact with DNA in a sequence-specific manner. Chimeras of [Rh(phi)2(bpy')]³⁺ (bpy' = 4-(4-carboxybutyl), 4'-methyl-2,2'bipyridine) and [Rh(phi)2(phen')]³⁺ (phen' = (5-amidoglutaryl)-1,10-phen-anthroline) and four different zinc finger peptides (Sp1 finger 2 and 3, ADR1b and ADR1b-Ala) have been successfully synthesized using solid phase coupling methodology. Electronic spectroscopy showed the rhodium complex and peptide to be essentially independent units. A method to successfully fold the peptide portion of the chimera with zinc has been developed, and ¹H HMR spectroscopy has been used to confirm folding. The resultant chimeras bind tightly to DNA, and the rhodium intercalator promotes DNA cleavage with photoactivation. Analysis of the DNA sites targeted by the chimeras on DNA restriction fragments have demonstrated that the peptide can direct new recognition. Variations in the rhodium complexes and peptides resulted in differences in specificity as seen by photocleavage. Studies on smaller oligonucleotides containing the recognition sequences have shown the rhodium - Sp1-2 chimera to bind with affinities of 10⁷-10⁸ M^-1 for its target sites. Hence, formation of rhodium(III) - zinc finger chimeras provide a route to establish high affinity DNA binding by a single zinc finger domain. At some sites, the rhodium complex and zinc finger appeared to bind independently to adjacent segments. For the [Rh(phi)₂(phen')]³⁺ - Sp1 - 2 chimeras, a strong high affinity site (K_a greater than or equal to 10⁸ M^-1) was observed, where it was postulated that the rhodium complex and zinc finger bind to the opposite strands of the GCG binding site in a cooperative fashion. These rhodium (III) - zinc finger chimeras represent a new route to examine the specific interactions of a single zinc finger with DNA in chemical detail and provide the basis to build a family of sequence-specific DNA binding molecules.

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