Microfluidic Selection of Aptamers

描述(申请人提供)：适配子是已被选择为对离散分子靶标具有高亲和力和选择性的单链DNA或RNA序列。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，在治疗和诊断应用方面引起了极大的兴趣。不幸的是，传统的适配子选择方案与膜蛋白不兼容。考虑到70%的治疗靶点是膜蛋白，这是有问题的。由于这些大的、高带电的大分子通过细胞膜的低效输送，适体的应用进一步受到限制。我们建议使用基于微量自由流动电泳(？FFE)的选择来分离膜蛋白的高亲和力适配子。与基于毛细管电泳法(CE)的选择相比，FFE提供了更高的吞吐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类和表面活性物质体系兼容，首次允许针对完全完整的膜结合靶标进行适体选择。最初的选择将使用SERCA作为测试目标，SERCA是一种具有良好特性的膜蛋白，在心脏病中具有重要意义。SERCA将被溶解在胶束、双胶束和SUV制剂中，以评估这些模型与FFE的相容性，并确定膜结构对适体选择的影响。FFE将被用来分离与参与受体介导的内吞作用(RME)的膜蛋白靶标具有亲和力的适体，包括TFR、LDLR和FR-a。假设与这些靶标有亲和力的适配子将有助于进入细胞。这一点将在各种细胞模型中得到证实。FFE将被用来分离与小窝蛋白介导的内吞作用(CvME)有关的神经鞘糖脂靶标具有亲和力的适配子。假设对特定糖鞘糖脂靶标具有亲和力的适体不仅有助于进入细胞，而且还将其货物运送到特定的细胞位置，进一步 提高生物利用度。对与RME或CvME相关的靶具有亲和力的适配子的传输特性将进行彻底的表征。将使用来自一系列组织类型的癌症和非癌症细胞系来评估细胞选择性。将使用共聚焦显微镜和单细胞器毛细管电泳法评估药物在细胞内的分布。将确定有效地输送治疗上重要的大分子类别的能力，包括蛋白质、siRNA和纳米颗粒。

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.