An HIV infection model is presented to direct the chemical refinement of triplex forming oligonucleotides (TFOs). The primary goal of this project is to enhance the binding constant and site specificity of TFO-duplex DNA binding with specific focus on enhancing the efficacy of the HIV38p class of TFO. The approach is to focus of the use of nucleoside homologues which can form stable base triplets at sites of TA or CG inversion by means of altered H-bonding potential (the xanthosine and formycin class) or by means of an altered backbone configuration (the stretched backbone class). To achieve this program of TFO enhancement, the investigator exploits an on-going collaboration between groups with expertise in molecular modeling, natural products and nucleic acid chemistry and nucleic acid biophysics. The second goal is to try to develop useful terminal modifications. Also, the author will try to enhance TFO stability with respect to cellular nucleases, to maximize the rate of TFO uptake into the nucleus and to explore the use of TFO-alkylator conjugates which can be covalently crosslinked to their duplex DNA target site. The investigator is particularly interested in these alkylator conjugates as tools to measure TFO binding in vivo and as a mechanism to enhance the efficacy of TFO-mediated transcription arrest. The duplex DNA binding potential of the proposed TFO homologues will be explored by structural and thermodynamic analyses employing UV spectroscopy, NMR, band shift and footprinting methods. The """"""""lead"""""""" HIV38p homologues which exhibit enhanced binding affinity, stability or cell uptake characteristics, or which have been shown to possess the capacity for efficient TFO-mediated crosslinking, will be assessed for enhanced antiviral activity in U937 and MT4 assays.
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