Analysis of the double strand content of DNA and the reaction of DNA with cis-dichlorodiammineplatinum(II)

Includes bibliographical references.Includes illustrations.Obtaining proton nuclear magnetic resonance (NMR) spectra with good resolution of large molecular weight, native, double stranded salmon testes DNA is possible, with some sample preparation. Spectra of large molecular weight DNA are not easily resolved by NMR, because molecular interactions with the aromatic protons of the nucleic bases produce chemical shifts of varying size. For the DNA samples to be similar to the DNA found in biological systems, the DNA was extensively treated with S₁ nuclease, an enzyme specific for single stranded DNA. To determine the extent of single stranded DNA present in the samples, a quantitative method to assay for the content of double stranded DNA was developed. The assay follows the standard addition method, and consists of incubating mixtures of denatured DNA and the sample DNA with S₁ nuclease. The fraction of single strands is determined from the ratio of mononucleotide products formed by the enzyme digestion, and the sample DNA present during the assay. The reaction between cis-dichlorodiammineplatinum(II) and DNA was studied using proton NMR. cis-Pt(NH₃)₂Cl₂ is an anti-tumor agent currently used in chemotherapy for genital- urinary cancers in man. The compound is known to interact with DNA in cells by inhibiting replication of DNA, leading to the death of the tumorous cell. Although this compound is approved as a drug, its products with DNA have never been positively identified. Shifts in the NMR spectra of the DNA nucleic base resonances were noticed after the addition of cis-Pt(NH₃)₂Cl₂.M.S. (Master of Science