Studies of catalytic nucleic acids targeted against the transcript of the PTEN tumor suppressor

Nucleic acid enzymes (NAE) are important tools in the down-regulation of RNA, either to study gene function and cellular pathways or potentially for therapeutic purposes. For an NAE to be effective in vivo, it needs to have an accessible binding site within the secondary structure of the target RNA, have good catalytic efficiency and be specific for the target. The purpose of this thesis was to address these factors through the design of efficient hammerhead ribozymes (hRz) and DNAzymes (Dz), targeting the PTEN tumor suppressor RNA.;Computer programs (based on thermodynamic assumptions) such as Mfold are commonly used to predict RNA secondary structure and thereby identify single-stranded accessible sites. Catalytic nucleic acids can also be designed using a number of in vitro selection strategies, including a SELEX-based method developed in our lab. In this study, a comparison was made of the cleavage efficiencies of hRz and Dz designed using Mfold, with the efficiencies of hRz and Dz designed using our in vitro selection technique. The highest level of activity obtained for both groups was attained using the library selection technique.;Two hRz (hRz 424 and hRz 680) and 3 Dz (Dz 278, Dz 422 and Dz 1063) developed from the selection were cloned into SNIP, a specialized self-processing hRz expression cassette, and transiently transfected into NIH3T3 mouse embryo fibroblasts. The three different Dz were modified with a 3′-3 ′ thymidine linkage to increase stability and were subsequently transfected into NIH3T3 cells. At specific time points following transfections RNA was isolated and analyzed for PTEN expression using the QPCR technique. In addition, immunocytochemistry was performed using a PTEN monoclonal antibody.;In spite of the good catalytic activity observed in vitro, the level of PTEN expression using either of the hRz did not appear to decrease appreciably in comparison to that of the control. However, Dz 278 reduced PTEN RNA in the NIH3T3 cell line. This decrease was accompanied by substantially decreased PTEN protein expression in a subpopulation of the cells. Thus, Dz 278 showed the highest levels of efficacy both in vitro and in vivo.