Date of Graduation

1998

Document Type

Dissertation/Thesis

Abstract

Triplex DNA is a unique structure formed by the sequence specific interaction of triplex forming oligonucleotides (TFOs) with DNA, via hydrogen bonds, in its major groove. For this binding to occur base triplets like G(ON)-G:C(DNA), T(ON)-A:T(DNA), C+(ON)-G:C(DNA) must form, subsequent to the interaction of the TFOs with the DNA. TFOs directed to bind to the promoter or to the internal region of the oncogene, can prevent its transcription, and ultimately abolish oncoprotein production. This study has investigated the use of a novel modified nucleoside analog OdA (8-oxo-2{dollar}\\sp\\prime{dollar}-deoxyadenosine) for targeting the G:C rich regions of DNA to form OdA-G:C triplets with the DNA. The efficacy of this binding has been determined using nuclear magnetic resonance spectroscopy, gel electrophoresis, band shift assays, in vitro binding assays (thermal denaturation curves T{dollar}\\sb{lcub}\\rm m{rcub}{dollar}) and association constant data. This novel analog exhibits enhanced binding to the G:C targets, compared to the other analogs being studied currently. The effect of OdA TFOs on the inhibition of c-Ha-ras-oncogene transcription, in T24 human bladder carcinoma cells, has also been studied. The use of receptor mediated uptake pathways, use of fusogenic peptides and cationic lipid formulations have achieved the cellular uptake and targeting of these TFOs. Evaluation of the efficacy of the TFOs and the various peptide and lipid formulations has been done by cell growth analysis and by the western blot analysis of the p21 ras oncoprotein.

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