Design, Synthesis and Characterization of Sequence Specific DNA Cleaving Agents (2013)

Chapter I: Synthetic, Sequence Specific DNA Cleaving Peptides Synthetic peptides based on the sequence specific DNA binding domain of Hin recombinase have been prepared which are equiped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. Covalent attachment of EDTA converts the sequence specific DNA binding pep tides into peptides which are capable of DNA strand scission in the presence of iron II, a reducing agent and molecular oxygen. The EDTA-equiped peptides cleave DNA at Hin binding sites and provide information on the nucleotide postion and groove identity of the modified amino acid residue when bound to DNA. The EDTA-peptides were also competent at DNA double strand cleavage, which is a useful characteristic for identifying binding sites on large DNA fragments (>5000 base pairs). A general procedure for the incorporation of an EDTA equiped lysine residue at any position in a synthetic peptide is also described. Results with 22 synthetic peptides indicate that Hin specifically recognizes a minor groove 5'-AAA sequence in its binding site with the peptide sequence Arg-Pro-Arg. Furthermore, the minor groove recognition event contributes substantially to the overall binding affinity. The possibility that minor groove recognition of tracks of A's by the peptide sequence Arg-Pro- Arg (and similar sequences), may be a common recognition motif in sequence specific DNA binding proteins is discussed. Chapter II: Distamycin-Actinomycin Hybrid Affinity Cleaving Molecules One approach to the design of sequence specific DNA binding molecules that read large sequences of double helical DNA is to couple DNA binding units of similar or diverse base pair specificities. Covalent attachment of his- and tris-N-methylpyrrolecarboxamides (based on the sequence specific DNA binding antibiotics Netropsin and Distamycin) and an aromatic phenoxazone intercalator (based on the sequence specific DNA intercalator Actinomycin) creates hybrid DNA binding minor groove and intercalation compounds. Using the affinity cleaving method we find that a first generation hybrid, bis-(EDTA-distamycin-glycyl)phenoxazone, binds the sequence 5'-TTATGGTTAA-3' which is consistent with simultaneous minor groove binding of the two tripyrrolecarboxamide units and intercalation of the phenoxazone moeity. Along with the targeted trimeric binding, the first generation hybrid also gave substantial monomeric/ dimeric binding, and exihibited only modest sequence specificity on large DNA fragments. In an attempt to reduce monomeric and dimeric binding, a second generation hybrid was prepared. Replacement of one pyrrolecarboxamide with a γ aminobutyric acid moeity, bis(EDTA-netropsin-glycyl)phenoxazone, generated a hybrid molecule which showed a reduced tendency for monomeric and dimeric DNA binding modes. The second generation hybrid binds to sequences of the form 5'-(A/T)₄GT(A/T)₄-3' and 5'-(A/T)₄CT(A/T)₄-3' and showed substantially improved sequence specificity on large DNA fragments relative to the first generation hybrid. Chapter III: Bleomycin-Distamycin Hybrid Affinity Cleaving Molecules Hybrids of the sequence specific DNA binding bithiazole domain of Bleomycin and tris-N-methylpyrrolecarboxamide of Distamycin are described. The hybrids address the issue of whether or not the bithiazole unit of the GC specific Bleomycin is a minor groove binding function. By the affinity cleaving method we find that hybrids in which the bithiazole is attached to either the amino or carboxy terminus of the tris-N-methylpyrrolecarboxamide, do not significantly alter the sequence specificity of the pyrrolecarboxamide domain. The results indicate that either the bithiazole unit is not a minor groove binding function in the same sense as the tripyrrolecarboxamide, or, alternatively, the bithiazole does not include all of the required sequence specific DNA recognition elements of Bleomycin. Chapter IV: Studies in Metalloporphyrin Mediated Affinity Cleaving Metalloporphyrins are known to cause DNA strand scission using a variety of central metals, and various reducing and oxidizing cofactors. To investigate if a metalloporphyrin can replace an EDTA•Fe chelate in an affinity cleaving molecule, a metalloporphyrin-Distamycin (PD) was prepared. PD•Fe showed very inefficient DNA cleavage compared to EDTA•Fe equiped affinity cleaving compounds. Additionally, PD•Fe showed no detectable sequence specific DNA cleaving ability. PD•Fe was also less efficient at DNA strand scission than ferriprotoporphyrin itself, which indicates that the DNA binding domain (tripyrrolecarboxamide) and cleaving domain (metalloporphyrin) are interfering with each other’s function in the affinity cleaving compounds.