Microfluidic Selection of Aptamers

描述（由申请人提供）：适配体是ssDNA或RNA序列，已被选中对离散分子靶标具有高亲和力和选择性。这些分子的亲和力和选择性，加上易于合成，低免疫原性和强大的化学稳定性，在治疗和诊断应用中产生了巨大的兴趣。不幸的是，传统的适体选择方案与膜蛋白不相容。考虑到70%的治疗靶点是膜蛋白，这是有问题的。适体的应用进一步限制了这些大的、高电荷的大分子在细胞膜上的低效递送。我们建议使用微自由流动电泳(？基于FFE)的选择分离高亲和力的膜蛋白适配体。？与基于毛细管电泳（CE）的选择相比，FFE提供了更高的通量和更简单的组分收集。最重要的是？FFE与用于溶解膜蛋白的脂质和表面活性剂系统兼容，首次允许对完全完整的膜结合靶标进行适体选择。初步选择将使用SERCA作为测试靶点，SERCA是一种具有良好表征的膜蛋白，在心脏病中具有重要意义。SERCA将溶于胶束、bicelle和SUV制剂中，以评估这些车型与？并确定膜结构对适体选择的影响。？FFE将用于分离与受体介导的内吞作用（RME）相关的膜蛋白靶点（包括TfR、LDLR和FR-a）有亲和力的适配体。据推测，对这些靶标具有亲和力的适体将促进进入细胞。这将在各种细胞模型中得到证实。？FFE将用于分离与小窝蛋白介导内吞作用（CvME）相关的鞘糖脂靶点有亲和力的适配体。据推测，具有特异性糖鞘脂靶亲和力的适体不仅能促进进入细胞，还能进一步将其货物运送到特定的细胞位置

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.