The long-term objective of this research is to improve the efficacy of the """"""""antisense"""""""" oligonucleotide approach to the control of virus replication with specific application to the HIV virus (AIDS). To achieve this aim, we will develope derivatives of oligonucleotides that are able to crosslink efficiently with target HIV RNAs in the environment of an infected cell. Cross-linking will be achieved by means of complexes of divalent platinum of type that are already known to attacks DNA in vivo. We will prepare the 5'-phosphorothioates of antisense oligonucleotides and treat them with reagents such as trans-[PtCl2(NH3)2] in order to obtain oligonucleotide adducts that include a reactive Pt-Cl bond at their 5'-termini. We will then carry out a systematic study of the cross-linking of these Pt- containing adducts to target RNAs of the HIV virus. We will choose the regions that are known to be sensitive to antisense inhibition as our first target sequences. In order to optimize cross-linking we will study systematically the way in which cross-linking efficiency depends on the detailed sequence of the target opposite the phosphorothioate group. We will also explore the effect of changes in the ligands attached to PtII on cross-linking Once precise targets are defined, we will extend our work to PtII adducts of non-hydrolyzable analogous of the oligodeoxynucleotide, such as methylphosphonates or phosphorothioates. As soon as we have identified efficient cross-linking reagents we will begin a collaboration with Douglas Richman, M.D. (Professor of Pathology and Medicine, University of California, San Diego, Departments of Pathology and Medicine, School of Medicine, Infectious Diseases Section (111F), Veterans Administration Medical Center, 3350 La Jolla Village Drive, San Diego, California, 92161) to study their effect on the replication of HIV virus in infected cells. Later in our program we will try to develop more complex oligonucleotide derivatives that enter cells more efficiently, for example derivatives carrying polylysine at the 3'-terminus as well as a reactive PtII complex at their 5'-end.
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