Microfluidic Selection of Aptamers

描述(申请人提供)：适配子是以高亲和力和选择性结合特定靶标的单链DNA或RNA。它们的高亲和力和选择性使它们成为优秀的候选药物或诊断试剂。使用被称为SELEX(通过指数富集法的配体的系统进化)的过程来分离适配子。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。使用过滤、层析或平移分离将与靶具有亲和力的序列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，为进一步的浓缩创造了一个新的池。经过8-15轮选择后，池中的相当一部分序列显示出与靶标的亲和力。适配子已被成功地用于小分子、多肽、蛋白质甚至整个细胞等广泛的靶点。不幸的是，分离适配子的过程仍然漫长而复杂。在前一个资金周期中，我们引入了毛细管电泳法(CE-SELEX)，它将分离适配子所需的时间从几周减少到几天。在目前提交的材料中，我们建议进一步利用微流控系统的优势，进一步简化和加快这一过程。我们还将为使用现有技术不容易进行的选择开发微流控方案。具体来说，我们建议：1)开发一种自动化的、流动的、微流控SELEX设备。我们将把微量FFE分离与流通式PCR和DNA纯化相结合，开发一种全自动微流控设备。我们预计，这款设备将能够在短短10分钟内进行一轮选择。2)建立了分离与膜蛋白有亲和力的适配子的CE-SELEX方法。到目前为止，绝大多数适配子已被选择与可溶性蛋白结合。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非常适合于对胶束或脂质体中的膜蛋白进行选择。3)建立具有催化活性的核酸适体分离新方法。目前已分离出数百种催化反应的适配子。不幸的是，由于用于分离这些适配子的选择过程，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检测和研究单个酶分子的技术作为一种新的方法的基础，该方法直接基于其催化活性来分离适体。

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.