Microfluidic Selection of Aptamers

描述（由申请人提供）：适配体是ssDNA或RNA，以高亲和力和选择性结合特定靶标。它们的高亲和力和选择性使它们成为极好的候选药物或诊断试剂。适配体的分离使用一个过程称为SELEX（配体的系统进化由指数富集）。在这个过程中，包含随机序列区域的核酸文库与感兴趣的目标孵育。对目标具有亲和性的序列通过过滤、层析或筛分分离从非结合序列中分离出来。这些结合序列被收集、扩增和纯化，产生一个新的库进行进一步的富集。经过8-15轮筛选后，池中相当一部分序列对目标具有亲和力。核酸适配体已经被成功地选择用于广泛的靶标，包括小分子、肽、蛋白质甚至整个细胞。不幸的是，分离适体的过程仍然漫长而复杂。在之前的融资周期中，我们引入了毛细管电泳选择（CE-SELEX），将分离适配体所需的时间从数周减少到数天。在目前的提交中，我们建议进一步利用微流体系统的优势，进一步简化和加快这一过程。我们还将开发微流体方案，以选择不容易使用现有技术进行。我们特别建议：1)开发一种自动化，流动，微流体SELEX装置。我们将把微ffe分离与PCR和DNA纯化相结合，开发一种全自动微流体装置。我们预计这台设备能在短短10分钟内完成一轮选择。2)建立CE-SELEX协议，分离具有膜蛋白亲和力的适体。迄今为止，绝大多数适体已被选择用于结合可溶性蛋白质。这是不幸的，因为膜蛋白的配体作为候选药物和诊断试剂更有价值。CE-SELEX非常适合对胶束或脂质体构成的膜蛋白进行选择。3)建立一种分离具有催化活性适配体的新方法。数百种催化反应的适体已经被分离出来。不幸的是，由于分离这些适体的选择过程，几乎所有这些核酸都直接参与反应，限制了它们的有效性。我们建议使用已开发的检测和研究单个酶分子的技术，作为直接根据其催化活性分离适配体的新方法的基础。

DESCRIPTION (provided by applicant): Aptamers are ssDNA or RNA that bind specific targets with high affinity and selectivity. Their combination of high affinity and selectivity make them excellent drug candidates or diagnostic agents. Aptamers are isolated using a process referred to as SELEX (Systematic Evolution of Ligands by Exponential enrichment). In this process a nucleic acid library containing a random sequence region is incubated with the target of interest. Sequences with affinity for the target are separated from non-binding sequences using filter, chromatography or panning separations. These binding sequences are collected, amplified and purified, generating a new pool for further rounds of enrichment. A significant fraction of sequences in the pool exhibit affinity for the target after 8-15 rounds of selection. Aptamers have been successfully selected for a wide range of targets including small molecules, peptides, proteins and even entire cells. Unfortunately the process for isolating aptamers remains long and complex. In the previous funding cycle we introduced capillary electrophoresis selections (CE-SELEX) which reduced the time required to isolated aptamers from weeks to days. In the current submission we propose to further leverage the advantages of microfluidic systems to further simplify and speed the process. We will also develop microfluidic protocols for selections that are not easily performed using existing techniques. In particular we propose to: 1) Develop an automated, flow through, microfluidic SELEX device. We will combine a micro-FFE separation with flow through PCR and DNA purification to develop a fully automated microfluidic device. We anticipate that this device will be able to perform a single round of selection in as little as 10 minutes. 2) Develop a CE-SELEX protocol for isolating aptamers with affinity for membrane proteins. To date the vast majority of aptamers have been selected to bind soluble proteins. This is unfortunate since ligands for membrane proteins are much more valuable as drug candidates and diagnostic agents. CE-SELEX is ideally suited to perform selections against membrane proteins constituted in micelles or liposomes. 3) Develop a new protocol for isolating aptamers with catalytic activity. Hundreds of aptamers that catalyze reactions have been isolated. Unfortunately, due to the selection process used for isolating these aptamers, almost all of these nucleic acids directly take part in the reaction, limiting their effectiveness. We propose using techniques developed to detect and study individual enzyme molecules as the basis for a new method that isolates aptamers based directly on their catalytic activity.