James R. Carter, James Dawson, Pruksa Nawtaisong, Velmaburgen Balaraman, Tresa S. Fraser and Malcolm J. Fraser, Jr.
Scientific Tracks Abstracts: JAA
Traditional viral detection methods can take hours to days before results may be obtained for an infected patient. We are developing a novel, rapid Dengue virus (DENV) diagnostic approach using an aptazyme complex consisting of a previously described anti Dengue virus hammerhead ribozyme and the theophylline-sensing aptamer (theoD-hRAz). Hammerhead ribozymes (hRz) have been used to target a number of RNA viruses, including HCV and HIV-1, in a sequence specifi c manner without the need for native cellular cofactors. RNA aptamers, RNA-based ligand binding molecules, have been generated to bind a wide range of ligands with high affi nity and selectivity. Targeting of the DENV-2 NGC genome resulted in a conformational change of the module that connects the hRz to the theophylline aptamer. Th is conformational change leads to an alteration in aptamer structure allowing for binding of theophylline resulting in a purple to clear color transition of the reacted sample indicating detection of DENV RNA. In vitro cleavage analysis of in vitro transcribed theoD-hRAz against Dengue virus type 2 New Guinea strain (DENV-2 NGC) showed the hRz portion of the aptazyme retains targeting and catalytic activities in the presence of an appended theophylline-specifi c aptazyme. Th e presence of theoD-hRAz RNA is confi rmed by RT-PCR. Th e theophylline aptamer has a published Kd<1μM. Equilibrium-fi ltration experiments, used to determine aptamer-ligand affi nity, showed the theophylline aptamer was able to eff ectively and specifi cally bind the ligand, theophylline, while appended to the hRz. Viral RNAs isolated from Vero cells infected with DENV-2 NGC at increasing M.O.I, were titrated into a reaction mixture containing theoD-hRAz conjugated to agarose beads to establish the utility and limits of specifi city of our engineered theoD-hRAz. RT-PCR analysis confi rmed the presence of DENV in the reaction mixture. Th ese results validate a unique approach towards coupling eff ective DENV targeting with the ligand binding capabilities of an RNA aptamer for the rapid detection of viral RNAs.
