(587a) In Vivo Monitoring of Viral Proteolytic Activity Using Luminescent Quantum Dot FRET-Based Probes

Proteases are enzymes that hydrolyze the peptide bonds of proteins. Many infectious microorganisms like bacteria and viruses use proteases as the key virulence factor for infection. Therefore proteases serve as an important target for characterization of disease development and can be potentially used to study various therapeutic agents. Here we are focusing on the use of a principle called fluorescence resonance energy transfer (FRET) to monitor the proteolytic activity of viruses in vivo. In particular, we seek to demonstrate the use of quantum dot bioconjugates to serve as sensitive probes in detecting viral protease activity in mammalian cells. Most commercially available quantum dots are not suitable for biological applications because of the presence of hydrophobic capping ligands on their surfaces. However, this can be overcome by exchanging the native TOPO capping with another surface ligand DHLA (dihydrolipoic acid), which renders them water soluble. In this work, the luminescent probes are based on FRET between a quantum dot that serves as an energy donor and rhodamine (an organic dye) that behaves as an energy acceptor. For the initial testing, we developed a substrate peptide sequence that contains the cleavage site which is recognized by the polio viral protease PV2A^pro. The rhodamine molecules are immobilized onto the surface of the quantum dots through the substrate peptide structure. The substrate peptide sequence acts as a spacer between the quantum dot and rhodamine thereby providing an optimal spacing for FRET to occur. Since our goal is to detect the presence of viral protease in living cells, a method that enables efficient intracellular delivery based on the use of the TAT peptide, which is derived from HIV-1 protein and has been shown to serve as excellent target carriers, was used. In this manner, the probes were introduced into BGMK (Buffalo green monkey kidney) cells and infected with different viral dosages. The linkage between the quantum dot donor and the rhodamine acceptor is cleaved during viral infection, due to the production of 2A^pro causing changes in fluorescence intensity. Utility of these new probes for monitoring viral activity and to screen for protease inhibitors will be discussed.