Antisense and triplex DNA (tx DNA) based pharmaceuticals offer an exciting approach to treat gene-based diseases with the potential for high selectivity and low toxicity. The molecular basis for these technologies offers new tools in molecular biology. At the molecular level, antisense and tx DNA agents act by binding to mRNA and DNA, respectively, and thereby inhibit gene expression. These agents have been extensively studied by spectroscopic methods in relatively simple chemical systems. However, the spectroscopic methods currently used have not been applied to more complicated and biologically relevant systems due to current sensitivity and resolution limitations. Therefore, more indirect and inconclusive methods have been used to study the activity of antisense and tx DNA oligonucleotides (ONs) in cellular systems, severely limiting the current understanding of these agents. The development of antisense and tx DNA agents would be facilitated if a more robust spectroscopic method were available for their study in cellular systems. A method that has been used to study biochemical processes in cellular systems is the spin probe labeling method. This method utilizes a substrate suitable labeled with a spin probe that can be monitored by ESR and is quite sensitive and selective. Here the investigators propose to extend the spin probe method by applying it to the study of antisense and tx DNA based pharmaceutical agents. ONs will be prepared that are spin probe labeled on a base or on a DNA intercalator covalently bonded to the end of the ON. The modified ONs will be compared with the corresponding unmodified ONs by thermal denaturation and gel electrophoresis methods. The ESR signatures unique to these spin probe labeled ONs in double stranded DNA (ds DNA) or tx DNAs (base or intercalator spin labeled) will be determined. The ONs will also be used to determine association constants for tx DNA formation. There are few methods available for measuring association constant formation for tx DNA and this is an important quantity to measure since it reflects the effectiveness of binding to the target. Finally, the investigators will examine the formation of tx DNA in a cellular system. CV-1 cells infected with SV40 virus will be treated with an ON designed to bind to the SV40 DNA, forming a tx DNA. The process will be monitored by ESR for tx DNA formation, assayed for their cytopathic effects and the ESR data correlated with the cytopathic effect data. The development of the ESR spin probe technique for studying antisense and tx DNA will significantly aid the development of these agents by providing a tool to monitor their binding to their cellular targets. Moreover, these agents may also be used in the future to study other aspects of these agents including cellular uptake and concentration in the nucleus. Likewise, by extension of the studies proposed here, these agents may be useful for studying other novel DNA structures in cells such as telomeric DNA.
