DNA Cross-Linking with Metallointercalator-Peptide Conjugates

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• 作者：Kimberly D. Copeland ; Alexis M. K. Lueras ; Eric D. A. Stemp ; and Jacqueline K. Barton
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 22, 2002
• 年：2002
• 卷：41
• 期：42
• 页码：12785 - 12797
• 全文大小：270K
• 年卷期：v.41,no.42(October 22, 2002)
• ISSN：1520-4995

文摘

Short peptides have been tethered to a DNA-intercalating ruthenium complex to create aphotoactivated cross-linking reagent. The ruthenium complex, [Ru(phen)(bpy')(dppz)]²⁺ (phen = 1,10-phenanthroline, bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine, and dppz = dipyridophenazine), deliversthe peptide to DNA and initiates the cross-linking reaction by oxidizing DNA upon irradiation in thepresence of an oxidative quencher. The tethered peptide, only five to six residues in length, forms cross-links with the oxidized site in DNA. Cross-linking was detected and studied by gel electrophoresis andthrough spectroscopic measurements. The ruthenium-peptide complex is luminescent when bound toDNA, and the binding constants for several intercalator-peptide conjugates were determined byluminescence titration. The composition of the peptide affects both binding affinity and the extent ofcross-linking. The greatest amounts of cross-linking were observed with tethered peptides that containedpositively charged residues, either lysine or arginine. To test the impact of individual residues on cross-linking, the central residue in a 5-mer peptide was substituted with seven different amino acids. Thoughmutation of this position had only a small effect on the extent of cross-linking, it was discovered thatpeptides containing Trp or Tyr gave a distinctive pattern of products in gels. In experiments using theuntethered peptide and ruthenium complex, it was determined that delivery of the peptide by the rutheniumintercalator is not essential for cross-linking; peptide attachment to the metal complex can constrain cross-linking. Importantly, the cross-linking adducts produced with ruthenium-peptide conjugates are luminescentand thus provide a luminescent cross-linking probe for DNA.