Development of High Throughput Aptamer-based Protein Capture/Detection Assays tow

描述（由申请人提供）：需要新型蛋白质捕获和检测试剂，以全面了解蛋白质组在基本生物过程和人类健康和疾病中的相互作用。这些试剂需要对特定的蛋白质靶点具有高亲和力和特异性，并且它们需要易于创建和生产，并且可以进行修饰和固定化，以便进行高通量的蛋白质分析。抗体是最常用的蛋白质捕获试剂，具有很高的亲和力和特异性；然而，由于它们的蛋白质性质，它们难以大规模生产和在高通量蛋白质捕获/检测分析中实施。单链寡核苷酸（适体）已成为替代的蛋白质捕获试剂。通过称为SELEX的迭代体外过程，可以从包含多达1013-1015个单个分子的大型随机序列寡核苷酸池中选择特异性结合靶蛋白的适体。该项目背后的总体假设是，SELEX过程可以自动化和多路复用，从而可以同时选择许多蛋白质的适体，所选的适体可以用于高通量分析，从而可以分析生物和医学样品中的目标蛋白质。为此，我们选择了100多个靶蛋白作为初始靶蛋白集，对具有不同生化特性和亚细胞定位、不同剪接变异体和翻译后修饰的蛋白进行方案测试。此外，这组代表了医学相关蛋白质的光谱。两种互补的SELEX策略将用于适配体选择，其中微流控装置将蛋白质保存在微阵列液体玻璃（溶胶-凝胶）液滴或完整的酵母细胞中，在其表面显示表达的人类蛋白质。选定的适体池将使用大规模平行测序技术进行测序，合成后将克隆单个适体的模板。在验证一对适体与单个靶蛋白的非竞争性结合后，这些适体对将用于高通量三明治分析。这些检测将在项目早期使用现有的蛋白质特异性适配体及其靶蛋白的融合体进行测试和优化。最后，将这些新的蛋白质捕获/检测试剂和分析方法与其他试剂和分析方法（如ELISA中的抗体）进行比较。预计该项目将对基础生命科学研究和医学研究产生重大影响。这项技术的发展将有助于选择其他重要的生物学和医学蛋白质的适体，所选的适体和用它们开发的分析可以立即应用于分子治疗和疾病诊断。

DESCRIPTION (provided by applicant): Novel protein capture and detection reagents are required to understand comprehensively the interplay of the proteome in basic biological processes and in human health and disease. These reagents need to have high affinity and specificity for particular protein targets, and they need to be easily created and produced, and be amenable to modification and immobilization for high throughput analysis of proteins. Antibodies, the most commonly used protein capture reagents, have high affinity and specificity; however, they are difficult to mass produce and to implement in high throughput protein capture/detection assays due to their protein nature. Single-stranded oligonucleotides (aptamers) have emerged as alternative protein capture reagents. Aptamers that specifically bind to a target protein can be selected from large random-sequence oligonucleotide pools containing as many as 1013-1015 individual molecules by an iterative in vitro process called SELEX. The overarching hypothesis behind this project is that, the SELEX process can be automated and multiplexed to enable simultaneous selection of aptamers to many proteins, and the selected aptamers can be employed in high throughput assays that allow analysis of the target proteins in biological and medical samples. To this end, over 100 target proteins have been chosen as the initial target protein set to test the protocols with proteins having different biochemical properties and subcellular localizations, as wells as with different splicing variants and post-translational modifications. Additionally, this set represents a spectrum of medically relevant proteins. Two complementary SELEX strategies, where a microfluidic device that holds proteins in microarrayed liquid glass (sol-gel) droplets or intact yeast cells that display expressed human proteins on their surface, will be utilized in aptamer selections. Pools of selected aptamers will be sequenced using a massively parallel sequencing technology and templates of individual aptamers will be cloned after synthesis. After validation of non-competitive binding of a pair of aptamer to individual target proteins, these aptamer pairs will be utilized in high throughput sandwich assays. These assays will be tested and optimized earlier in the project using existing protein-specific aptamers and fusions of their target proteins. Finally, these new protein capture/detection reagents and assays will be compared to other reagents and assays, such as antibodies in ELISA. This project is expected to have a major impact on both basic life sciences research and medical research. The technological development will facilitate the selection of aptamers to other biologically and medically important proteins and the selected aptamers and the assays developed with them may have immediate applications in molecular therapeutics and disease diagnosis. PUBLIC HEALTH RELEVANCE: This project seeks to transform an existing technology to facilitate and streamline identification of novel capture reagents for human proteins and to incorporate these new reagents in assays that can accommodate simultaneous analysis of many proteins. This project will not only develop new technology and assays that enable analysis of human proteins critical in human health and disease, but also generate the novel reagents that can be used for therapy.