Microfluidic Selection of Aptamers

DESCRIPTION (provided by applicant): Aptamers are ssDNA or RNA sequences that have been selected to have high affinity and selectivity for discrete molecular targets. The affinity an selectivity of these molecules combined with ease of synthesis, low immunogenicity and robust chemical stability have generated tremendous interest in both therapeutic and diagnostic applications. Unfortunately, conventional aptamer selection protocols are incompatible with membrane proteins. This is problematic considering that >70% of all therapeutic targets are membrane proteins. Application of aptamers is further limited by inefficient delivery of these large, highly charged macromolecules across cell membranes. We propose using micro free flow electrophoresis (?FFE) based selections to isolate high affinity aptamers for membrane proteins. ?FFE provides higher throughput and simpler fraction collection than capillary electrophoresis (CE) based selections. Most importantly, ?FFE is compatible with the lipid and surfactant systems used to solubilize membrane proteins, allowing aptamer selections to be performed against fully intact membrane bound targets for the first time. Initial selections will b performed using SERCA, a well characterized membrane protein with important implications in heart disease, as a test target. SERCA will be solubilized in micelle, bicelle and SUV preparations to assess the compatibility of these models with ?FFE and determine the effect of the membrane structure on aptamer selection. ?FFE will be used to isolate aptamers with affinity for membrane protein targets involved in receptor mediated endocytosis (RME) including TfR, LDLR and FR-a. It is hypothesized that aptamers with affinity for these targets will facilitat entry into the cell. This will be confirmed in various cell models. ?FFE will be used to isolate aptamers with affinity for glycosphingolipid targets implicated in caveolin mediated endocytosis (CvME). It is hypothesized that aptamers with affinity for specific glycosphingolipid targets will not only facilitate entry into the cell but deliver their cargo to specific cell locations, further enhancing bioavailability. The transport characteristics of aptamers with affinity for targets associated with RME or CvME will be thoroughly characterized. Cell selectivity will be assessed using both cancerous and non- cancerous cell lines originating from a range of tissue types. Intracellular distribution of delivery will be assessed using confocal microscopy and single organelle capillary electrophoresis analysis. Ability to efficiently deliver therapeutically importnt classes of macromolecules, including proteins, siRNA and nanoparticles, will be determined.