HYDROLASE STABILITY ENHANCEMENT AND ITS APPLICATION TO SIRNA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Enzymes play an essential role in biotechnology where they are used to catalyze a broad range of biorelevant reactions.However enzymes exhibit poor stability, that is, their catalytic activity decreases drastically with prolonged exposure to aqueous or organic solvents. This limitation and the poor understanding of the relationship between stability, structure,and function severely limits the potential of enzymes. The main objectives of this research are to fully understand and enhance enzyme stability. Our studies show that poor enzyme stability is inherently due to minute but critical changes that occur in the active site during solvent exposure. Experimental and theoretical studies will determine the nature of these critical changes and establish causal relationships between these changes and enzyme stability. Novel methodologies will be developed to reduce the changes with in the active site during prolonged solvent exposure in order to enhance enzyme stability. The technical expertise and understanding gained in the study of enzyme stability will be applied to surpass a similar limitations faced by siRNAs. Short interfering RNAs are short double-stranded nucleic acids that are being developed to target therapeuticaly important genes in cancer, viral infections, and other diseases. These siRNA will be chemically modified with a variety of macromolecules to increase their stability and efficiency, and to increase their ability to cross a cell wall membrane. New techniques will be designed to directly measure siRNA stability in complex biological fluids. Proposed Specific Aims (SA): SA1: Study the enzyme's operational stability in terms of the role of the enzyme reversibly bound water and enzyme dynamics. The exchange of reversibly bound water molecules with the bulk of an organic solvent will be studied using nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). The effect of long term enzyme incubation in an organic solvent as well as the effect of organic solvent hydration on enzyme polarity will be measured using EPR. Molecular dynamics simulation of an EPR spin-label and of the reversibly bound waters and solvent molecules near the active site will complement the experimental study. SA2: Enhance the stability of enzymes in organic solvents by immobilizing the enzyme on polar and hydrophilic surfaces of materials such as nanosilicates, to ensure proper enzyme hydration and stability. Enzyme stability determination on different surfaces will show correlation or causation between stability and overall hydration state of the enzyme. Molecular and quantum-mechanical dynamics simulation of the solvent-enzyme-material interface will suggest a hydration mechanism regarding surface water molecule movementand clustering. SA3: Modify systematically and rationally a pre-designed siRNA by co-lyophilization and chemical modification with a variety of macromolecules such as methoxy poly(ethylene glycol) (PEG) and cyclodextrins. HeLa cells will be transfected with these modified siRNA to knockdown a specific gene expression and evaluate possible increase to their stability, efficiency, and ability to cross the cell wall membrane. This study will provide physical and chemical insights into the mechanisms of siRNA stability and cell membrane transfer. Molecular dynamics simulation will be used to determine structural differences between modified and natural siRNA. SA4: Design new techniques involving our expertise with Forster resonance energy transfer (FRET), EPR and fluorine NMR to directly measure single-strand and duplex siRNA nucleolytic stability in complex biological fluids such as blood plasma, extra- cellular matrix components and cellular cytoplasm.