Novel combinatorial and structure-based design methods will be used to develop phosphorothioate and phosphorodithioate DNA decoys or aptamers as targeted therapeutics towards the human immunodeficiency virus (HIV). Development of these anti-AIDS agents will be facilitated by nuclear magnetic resonance (NMR) spectroscopy and computational biochemistry of both agent and protein agent complexes. We will specifically synthesize thioated backbone aptamer oligonucleotide analogues targeted to HIV-1 reverse transcriptase (RT) and nucleocapsid (NCp7) and the human transcription factor NF-kappaB. We have recently developed a novel combinatorial selection scheme for phosphorothioate hybrid backbone aptamers targeting the nuclear factor for human IL6 (NF-IL6), a transcription factor involved in the induction of acute-phase responsive and cytokine gene promoters in response to inflammation. Using a random combinatorial selection approach and dNTP(alpha)S s in PCR amplification, we have selected specific thio-substituted agents which have the highest specificity in binding (nM range) to NF-IL6. This is currently being extended to NF-kappaB and will also be applied to NCp7 and RT. A split synthesis scheme will be developed for combinatorial selection of dithiophosphate aptamers for these proteins. Since phosphorothioate and phosphorodithioate substituted oligonucleotides show reduced nuclease activity, these combinatorial thiophosphate-selection experiments can offer wide application for rapid identification of new therapeutic agents. This technology will allow us to develop separate aptamers targeting in principle any one of the 15 possible combinations of 5 homo- and heterodimers of the 5 different forms of NF-kappaB/Rel. NF-kappaB/Rel transcription factors, are key mediators of the immune and acute phase responses, apoptosis, cell proliferation and differentiation, and are key transactivators acting on the LTR of HIV-1. They thus represent potential therapeutic targets for control of HIV-1 proliferation. NMR will be used to define the three-dimensional structure of monothio- and dithiophosphate modified oligonucleotide agents and aptamer NCp7 complexes. We will also assess the in vivo activity of the aptamers in tissue culture testing to arrest HIV-1 proliferation and gene expression as well as to activate HIV gene expression to identify hidden reservoirs of infection. Finally, we will explore the feasibility of utilizing these highly selective thioaptamers for recognition of protein-protein interactions using a new DNA/protein chip technology for genetic analysis at the level of functional protein expression.
| Thiviyanathan, Varatharasa; Gorenstein, David G (2012) Aptamers and the next generation of diagnostic reagents. Proteomics Clin Appl 6:563-73 |
| Mann, Aman P; Bhavane, Rohan C; Somasunderam, Anoma et al. (2011) Thioaptamer conjugated liposomes for tumor vasculature targeting. Oncotarget 2:298-304 |
| Yang, Xianbin; Li, Na; Gorenstein, David G (2011) Strategies for the discovery of therapeutic aptamers. Expert Opin Drug Discov 6:75-87 |
| Kang, Jonghoon; Lee, Myung Soog; Copland 3rd, John A et al. (2008) Combinatorial selection of a single stranded DNA thioaptamer targeting TGF-beta1 protein. Bioorg Med Chem Lett 18:1835-9 |
| Fennewald, Susan M; Scott, Erin P; Zhang, Lihong et al. (2007) Thioaptamer decoy targeting of AP-1 proteins influences cytokine expression and the outcome of arenavirus infections. J Gen Virol 88:981-90 |
| Kang, Jonghoon; Lee, Myung Soog; Watowich, Stanley J et al. (2007) Combinatorial selection of a RNA thioaptamer that binds to Venezuelan equine encephalitis virus capsid protein. FEBS Lett 581:2497-502 |
| Kang, Jonghoon; Lee, Myung Soog; Gorenstein, David G (2007) Magnesium ion is an effective inhibitor of the binding of Escherichia coli to heparin. J Microbiol Methods 71:340-2 |
| Kang, Jonghoon; Lee, Myung Soog; Gorenstein, David G (2007) Application of RNase in the purification of RNA-binding proteins. Anal Biochem 365:147-8 |
| Kang, Jonghoon; Lee, Myung Soog; Gorenstein, David G (2007) Characterization of heparin-living bacteria interactions by chemiluminescence electrophoretic mobility shift assay. Anal Biochem 363:312-4 |
| Bowick, Gavin C; Fennewald, Susan M; Elsom, Barry L et al. (2006) Differential signaling networks induced by mild and lethal hemorrhagic fever virus infections. J Virol 80:10248-52 |
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