P04.03 * GLIOMA SPECIFIC TARGET IDENTIFICATION WITH APTAMERS

Abstract

DESIGN: Aptamers are in vitro generated, short DNA and RNA sequences which are randomly created as a library. They are created with multiple permutations and combinations automatically within set primer ends. These are then exposed to the target structure against which we want an aptamer ‘selected’, using Sequential Enumeration of Ligands by Exponential enrichment (SELEX). Modified aptamers based on published sequences against glioma cell lines and newly generated sequences were used in the project to identify their binding targets. SUBJECTS: Commercially available glioma and glial cell lines were used along with in-house generated primary glioma cultures. METHODS: Cy3 and biotin- conjugated aptamers were incubated with live glioma cell cultures and imaged using confocal or light microscopy. This established proof of concept that the aptamers were binding to the glial cell more specifically than control. To determine the target ligand, aptamers were then reacted with glial cell lysate and subjected to precipitation using streptavidin agarose beads and SDS polyacrylamide gel electrophoresis. Proteins were then analysed by mass spectroscopy. RESULTS: Known and unknown aptamer protein ligands were co-precipitated. X ray cross complementing protein 6 (XRCC6 or Ku70) and x ray cross complementing protein 5 (XRCC5 or Ku80) were precipitated along with nucleolin and other related proteins. Heterogenous nuclear ribonuclear proteins or hnRNP's were also isolated. Ku70 and Ku80 were also precipitated in primary cell cultures. KU70 and Ku80 appear in the KEGG pathway and Non-homologus end joining pathway or NHEJ for DNA repair. CONCLUSIONS: The aptamer has shown preferential binding to glioma cells and could act as a delivery system for therapeutic payloads. The aptamer targets Ku70 and Ku80, which are known to be over expressed in other forms of cancer but their role in gliomagenesis has not been fully elucidated. Other novel proteins have also been identified. Thus the aptamer co-precipitation technique has identified potential glioma biomarkers that may be of clinical significance.