Microfluidic Selection of Aptamers

描述（由申请人提供）：适体是以高亲和力和选择性结合特异性靶标的ssDNA或RNA。它们的高亲和力和选择性的组合使它们成为优秀的候选药物或诊断剂。使用称为SELEX（通过指数富集的配体系统进化）的方法分离适体。在该过程中，将含有随机序列区域的核酸文库与感兴趣的靶标一起孵育。使用过滤、色谱或淘选分离将对靶具有亲和力的序列与非结合序列分离。收集、扩增和纯化这些结合序列，产生新的库用于进一步的富集。在8-15轮选择后，池中的显著部分的序列表现出对靶的亲和力。适体已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。在上一个资助周期中，我们引入了毛细管电泳选择（CE-SELEX），将分离适体所需的时间从数周缩短到数天。在当前提交的文件中，我们建议进一步利用微流体系统的优势，以进一步简化和加速该过程。我们还将开发微流体方案，用于使用现有技术不容易进行的选择。具体而言，我们提出：1）开发自动化的、流通的、微流体SELEX装置。我们将联合收割机结合微FFE分离与流通PCR和DNA纯化，以开发全自动微流控装置。我们预计，该设备将能够在短短10分钟内执行一轮选择。2)开发用于分离对膜蛋白具有亲和力的适体的CE-SELEX方案。迄今为止，绝大多数适体已被选择为结合可溶性蛋白质。这是不幸的，因为膜蛋白的配体作为候选药物和诊断剂更有价值。CE-SELEX非常适合对胶束或脂质体中的膜蛋白进行选择。3)开发新的分离具有催化活性的适体的方案。已经分离出数百种催化反应的适体。不幸的是，由于用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。我们建议使用开发的技术来检测和研究单个酶分子作为一种新方法的基础，该方法直接基于其催化活性分离适体。

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.