Abstract
INTRODUCTION: Aptamers are in vitro generated DNA and RNA sequences. Aptamer sequences have multiple permutations and combinations of nucleotides and are then ‘selected’, using Sequential Enumeration of Ligands by Exponential Enrichment (SELEX) towards the desired, known or unknown target. Modified aptamers based on published sequences against glioma cell lines and newly generated sequences were used in the project to identify and confirm the targets. METHODS: Commercially available glioma cell lines and short term cultures from anonymised patient tissue were used. Fluorescent and biotin conjugated aptamers were incubated with glioma cell cultures and imaged using confocal or light microscopy. Aptamers were then reacted with glial cell lysate and subjected to precipitation using streptavidin agarose beads and SDS polyacrylamide electrophoresis. Proteins were analysed by mass spectroscopy. Ku 70 (X-ray Repair Cross Complementing protein 6), and Ku80 (X-ray Repair Cross Complementing protein 5) were identified as the most abundant protein when compared to control aptamer precipitation. Western blot analysis and siRNA knockdown was used to further confirm the target. RESULTS: Ku70 and Ku80 were precipitated along with known proteins associated proteins such as nucleolin as well as a few unknown proteins. Western blot analysis confirmed abundant precipitation of Ku70 and Ku80 when compared to control aptamer. DAB staining of cell cultures revealed over-expression of Ku70 and Ku80 throughout the cell. Knock down with siRNA showed reduced binding of the aptamer to the nucleus when compared to untreated cell cultures. CONCLUSIONS: Certain aptamers appear to be targeting DNA repair proteins Ku70 and Ku80 and could be used for developing targeted therapy towards glioma. Western and knockdown analysis, confirm the specificity of binding and leads the way for in-vivo testing of aptamers.