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Date of Award
Christine S. Chow
Cis-diamminodichloridoplatinum (II), cisplatin, is an antitumor drug that has been used to treat several types of cancers. The reaction of cisplatin with DNA has been studied and discussed extensively in the literature; however, the effects of cisplatin on RNA function are poorly understood. In this thesis, two aspects of cisplatin, its preferred sites of interaction with RNA and its use as a chemical probe to gain accessibility information, were explored.
To understand the site-selectivity of cisplatin with RNA, model RNA constructs and full-length 16S rRNA were employed. The binding studies revealed a cisplatin preference for guanosine-rich sequences. Primer extensions in 16S rRNA and MALDI-TOF in model constructs were used to locate the binding sites of cisplatin. HPLC and LC-MS were useful to determine the types and ratios of various adducts formed. Cisplatin and its analogues were employed to probe the accessibility of nucleotides on 16S rRNA, 30S subunits and 70S ribosomes in vitro as well as in vivo. This study revealed that many functionally important sites, such as helix 18, 24, 27, and 34 are accessible to the aquated platinum complex. Thus, these accessible sites can potentially be utilized as a new target sites in the design of structure-based antibiotics. When charge and size of the complex were changed, the binding preference was altered. In addition to the expected consecutive Gs, cisplatin analogues preferentially targeted AG sites on loop or bulge regions. Thus, several new complexes could be synthesized and utilized to gain more information about drug accessibility on the ribosome.
The last part of the research focused on the application of siRNA to target non-Hodgkin's lymphoma (NHL). Small interfering RNAs were designed to downregulate the c-Myc expression in NHL cells. Stabilities of designed siRNAs in media and their incorporation into liposomes were studied. Complexes of siRNA, liposomes, and antibody fragments (scFv) could be utilized in future applications to target specifically the c-Myc expression in NHL cells.
Overall, this thesis work explored cisplatin binding to RNA and a number of possible new antibiotic target sites on the ribosome were identified. In the long term, further studies with fully functional ribosomes and comparisons with other organisms will have a greater impact on identifying novel drug target sites in pathogenic bacteria.
Rijal, Keshab, "Exploring Potential Drug Target Sites In The Ribosome Using Cisplatin And Its Analogues" (2011). Wayne State University Dissertations. Paper 183.