Microfluidic Selection of Aptamers

描述（由申请人提供）：适体是已选择的对离散分子靶标具有高亲和力和选择性的ssDNA或RNA序列。这些分子的亲和力和选择性与易于合成、低免疫原性和稳健的化学稳定性相结合，在治疗和诊断应用中产生了巨大的兴趣。不幸的是，常规的适体选择方案与膜蛋白不相容。考虑到>70%的所有治疗靶标是膜蛋白，这是有问题的。适体的应用还受到这些大的、高电荷的大分子穿过细胞膜的低效递送的限制。我们建议使用微自由流电泳（？FFE）的选择以分离膜蛋白的高亲和力适体。?与基于毛细管电泳（CE）的选择相比，FFE提供更高的通量和更简单的级分收集。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相容，首次允许针对完全完整的膜结合靶标进行适体选择。初始选择将使用SERCA（一种在心脏病中具有重要意义的良好表征的膜蛋白）作为测试靶标进行B。SERCA将溶解在胶束，bicelle和SUV制剂，以评估这些模型的兼容性？FFE并确定膜结构对适体选择的影响。? FFE将用于分离对受体介导的内吞作用（RME）中涉及的膜蛋白靶标（包括TfR、LDLR和FR-α）具有亲和力的适体。假设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将用于分离对涉及小窝蛋白介导的内吞作用（CvME）的鞘糖脂靶标具有亲和力的适体。假设对特异性鞘糖脂靶标具有亲和力的适体将不仅促进进入细胞，而且将它们的货物递送到特定的细胞位置，进一步 提高生物利用度。将彻底表征对与RME或CvME相关的靶标具有亲和力的适体的转运特征。将使用源自一系列组织类型的癌性和非癌性细胞系来评估细胞选择性。将使用共聚焦显微镜和单个细胞器毛细管电泳分析来评估递送的细胞内分布。将确定有效递送治疗上重要类别的大分子（包括蛋白质、siRNA和纳米颗粒）的能力。 公共卫生相关性：该提案描述了用于选择对完整膜蛋白具有高亲和力的适体的新的微流体策略的发展。特别地，将靶向促进通过内吞作用进入细胞的膜结合受体。假设对这些靶标具有亲和力的适体将有效地递送蛋白质、siRNA、RNA、 和纳米颗粒到特定的细胞位置，大大提高了这些治疗上重要的大分子的生物利用度。

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. PUBLIC HEALTH RELEVANCE: This proposal describes the development of new microfluidic strategies for selecting aptamers with high affinity for intact membrane proteins. In particular, membrane bound receptors that facilitate entry into cells via endocytosis will be targeted. It is hypothesized that aptamers with affinity for these targets will efficiently deliver proteins, siRNA and nanoparticles to specific cell locations, dramatically improving the bioavailability of these therapeutically important macromolecules.