I. Bismethidium Intercalators: The Binding of Nucleic Acids and II. Experiments in the Design of Site Specific DNA Cleaving Agents

Citation

Ikeda, Richard Alan (1984) I. Bismethidium Intercalators: The Binding of Nucleic Acids and II. Experiments in the Design of Site Specific DNA Cleaving Agents. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/xp0y-6y73. https://resolver.caltech.edu/CaltechTHESIS:11122018-122020454

Abstract

CHAPTER I

Part I

Bis(methidium)spermidine (BMSpd), an analog of ethidium bromide in which two phenanthridine chromophores are tethered by a 10 Å Spermidine linker, has been synthesized and characterized in order to test the effect of linker length on DNA binding. Like bis(methidium)spermine (BMSp),¹ a dimer with a 15 Å tether, BMSpd unwinds DNA 36.5°. Unlike BMSp, the binding of BMSpd to calf thymus DNA, poly dG·poly dC, poly rA·poly dT, and poly dA·poly dT is complicated. At 0.4 M (M⁺) and pH 7, the binding site sizes and binding constants of BMSpd range from 2.75 base pairs (calf thymus DNA) to 4.0 base pairs (rA·dT, dG·dC) and 5 x 10⁵ M^-1 (dA·dT) to 6 x 10⁶M^-1 (r A·dT). These affinities are on the average, ten times higher than the analogous affinities of ethidium bromide and 300 times less than the binding constants of BMSp. The most dramatic change occurs in the binding specificity of BMSpd. Whereas, BMSp binds poly rA·dT 1440 times more tightly than poly dA·dT, the spermidine analog (BMSpd) binds poly rA·dT only 10 times more tightly than poly dA·dT.

Part II

The ethidium dimers, bis(methidium)spermine (BMSp) and bis(methidium)spermidine (BMSpd), were found to site selectively inhibit DNase I and a variety of restriction enzymes on the plasmid pBR322. In contrast to the dimers, the monomer, ethidium bromide (EB), shows no site selective action in competition with any of the enzymes tested. The sites preferentially inhibited by BMSp and BMSpd appear to be in the first third of the plasmid. These observations and the clustering of inhibited restriction sites then suggests that selective inhibition may be due to the recognition or perturbation of DNA secondary structure.

CHAPTER II

The compounds methidiumpropyl-EDTA, distamycin-EDTA, and pentaN-methylpyrrolecarboxamide-EDTA are three representative examples of a class of rationally designed DNA cleaving agents. By appending an iron chelator to different DNA binding vehicles certain properties of the resulting DNA cleaving agent can be controlled. Methidiumpropyl-EDTA randomly single strand cleaves DNA with high efficiency. Distamycin-EDTA nicks at 5 base pair recognition sites, and penta-N-methylpyrrolecarboxamide-EDTA double strand cleaves DNA at 6 base pair recognition sites. The success of these compounds led to the design of a controlled site cleaving agent, oligonucleotide-EDTA.

The construction of the molecule was approached in two ways, synthetically and enzymatically. Synthetically, the oligonucleotide-EDTA was constructed by known nucleotide triester methodology. Enzymatically, a modified uridine, EDTA-dUTP, was incorporated into a DNA fragment. Unfortunately, neither strategy yielded an active DNA cleaving agent. These failures, however, do not directly reflect on the viability of an oligonucleotide-EDTA. It is possible that an extended research effort will uncover the methods needed to create an active oligonucleotide-EDTA.

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