- Repository URL:
- chemistry.; Major chemistry.
thesis / dissertation description
The hammerhead ribozyme is a well-characterized motif that can catalyze the cleavage of a phosphodiester bond in a substrate-RNA backbone. This cleavage activity finds potential applications in ribozyme gene therapy by targeting the mRNA that code for proteins associated with diseases. Successful applications of this strategy have been demonstrated in treatment of AIDS, cancer and Alzheimer's disease. It is therefore of interest to be able to deliver the hammerhead ribozyme as a drug for therapeutic applications. PLGA (Poly-Lactide-co-Glyolide) is an FDA approved and extensively characterized biodegradable controlled release system for drug delivery. These copolymer systems are preferred over conventional modes as they release the drug at a constant rate and are more patient compliant. This work explores the drug delivery aspects of the hammerhead ribozyme from two kinds of PLGA formulations, which have a ratio of lactide to glycolide of 65:35 and 85:15. The encapsulation has been done by a pressure quench method using supercritical CO?, a technique not explored so far for nucleic acids. Studies done in this work reveal that the ribozyme can be encapsulated and released in its active form from a PLGA formulation, in a controlled fashion. However, it is observed that slowing down the rate of release is associated with greater degradation of RNA and a reduction in activity, particularly in a formulation with increased lactide content. These effects are attributed to the acidic pH generated in the polymer environment during degradation. The three-dimensional conformation of the hammerhead ribozyme is known to be critical for its activity. Electron Paramagnetic Resonance (EPR) spectroscopy using site-directed-spin-labeling (SDSL) of the ribozyme, with nitroxide labels covalently attached to the 5'-ends of stems I and III, has been explored as a tool for conformational analysis of the ribozyme, in view of extending the studies to probe the ribozyme for conformational changes in the polymer microenvironment. The results of these studies suggest that the distances between the 5'-ends of stems I and III are greater than 25 A as predicted by crystallographic studies. There is also evidence for the two-step folding mechanism predicted from fluorescence studies. It is expected that the two facets explored in the current work can contribute towards achieving an efficient drug delivery vehicle for the hammerhead ribozyme, and probing conformational changes related to its activity.