Programmable Multi-Target Detection Using an Aptamer-Integrated Nanopore

描述（由申请人提供）：该提案提出了一个跨学科的策略，将高灵敏度、高特异性适配体与基于纳米孔的单分子技术相结合。长期目标是开发用于生物医学检测的适配体集成纳米孔探针。适配体是一种工程化的DNA/RNA，可以特异性识别具有高亲和力的多种蛋白质。结合后，这些强大的分子可以抑制病原体蛋白，催化化学反应，控制基因表达，调节细胞功能。因此，适体可以作为探索生物系统的潜在工具。受这些小而复杂的分子的激励，我们希望将适体技术与我们的纳米孔探针结合起来，构建新一代的单分子探测器，这将极大地帮助各种疾病相关的检测。纳米孔技术可以通过结合时电导的离散变化，“直观”地捕捉到纳米级孔中单个分子与配体的动态结合。我们建议将纳米孔与实验室纳米制造、生物友好表面工程和分子工程相结合，以实现以下具体目标：了解和操作纳米孔的各种性质，以提高单分子检测；2. 建立了配体纳米孔单分子检测的基本模型。将测试具有不同适体类型（DNA/RNA）的广泛目标物种，包括免疫球蛋白E、HIV-1逆转录酶和大甲型流感病毒颗粒；3. 建立了采用工程化转移适体（t-Aptamer）介导检测的先进模型。t-适体由适体和通用载体组成，充当“翻译器”，将目标信息编码为载体和纳米孔中固定受体之间的杂交频率。这种t-适配体介导的检测是稳健的、可编程的，最终将导致一个通用的纳米孔，可以区分不同的目标并同时进行多目标检测。这项研究的成功将极大地扩展纳米孔作为生物医学分析和高通量筛选的新一代检测技术的能力。在纳米医学中，制造这样的工具对于活细胞的分子尺度成分（或纳米机械）的定量表征和精确控制具有重要意义。

DESCRIPTION (provided by applicant): This proposal addresses an interdisciplinary strategy that integrates the use of highly sensitive, highly specific aptamers with nanopore-based single molecule technology. The long term goal is to develop aptamer-integrated nanopore probes for biomedical detections. Aptamers are engineered DNA/RNA that can specifically recognize broad species of proteins with high affinities. Upon binding, these powerful molecules can inhibit pathogen protein, catalyze chemical reactions, control gene expression, and regulate cellular functions. Therefore aptamers can potentially be applied as tools for exploring biological systems. Motivated by such small, but sophisticated molecules, we would like to integrate aptamer technology with our nanopore probe to construct a new generation of single molecule detector that would aid greatly in diverse disease-related detections. Nanopore technology can "visually" capture the dynamic binding of a single molecule to a ligand in a nanometer-scaled pore through the discrete changes in conductance upon binding. We propose the combined use of nanopore with laboratory nanofabrication, bio-friendly surface engineering, and molecular engineering to accomplish the following specific aims: 1. Understand and manipulate various properties of nanopores for improvement of single molecule detection; 2. Establish a fundamental model for single molecule detection with aptamer-equipped nanopores. Broad target species with varied aptamer types (DNA/RNA) will be tested, including Immunoglobulin E, HIV-1 reverse transcriptase, and large influenza A viral particle; 3. Establishing an advanced model that employs engineered Transfer Aptamer (t-Aptamer) to mediate detection. The t-Aptamer, which composes an aptamer and a universal carrier, acts as a "translator" to encode target information into the frequency of hybridization between the carrier and the immobilized receptor in the nanopore. Such a t-Aptamer-mediated detection is robust, programmable, and will ultimately lead to a universal nanopore that can discriminate different targets and perform simultaneous multi-target detection. The success of this research will greatly expand the capability of nanopores as the new generation of detection technology for biomedical analysis and high-throughput screening. Fashioning such tools is significant in nanomedicine for the quantitative characterization and precise control of molecular scale components (or nanomachinery) of living cells.