Simple DNA/RNA Probes for Protein Targets

描述(由申请人提供)：我们描述了一种快速发现具有高亲和力和选择性地结合到感兴趣目标的小的、结构定义的核酸“探针”序列的综合方法。这些目标是蛋白质、生物恐怖因子、病原体和病毒。这些探头将被设计成专利的allSwitchTM指示器，该指示器对目标的存在做出快速反应，产生荧光或发光信号。 在这个第一阶段的项目中，我们建议快速筛选和分离高亲和力结合探针(c-探针)。我们将使用高通量方法从包含数十亿DNA序列的已定义文库中分离至少已知的针对α-凝血酶和血小板衍生生长因子(PDGF)的探针序列。将在具有预定义二级结构的文库中寻找凝血酶和PDGF的替代c-探针。 在第二阶段，这一办法将扩大到包括更多种类的结构主题和新目标。发现探针和创建allSwitch的目标将包括与凝血和血栓形成有关的蛋白质，以及与伤口愈合、血管生成和增殖性疾病有关的生长因子。基于allSwitch的分析将有助于发现小分子，这些小分子可用于调节血液中这些蛋白质的相互作用的治疗。这与基于适体或抗体的疗法形成了鲜明对比，后者很难防止血浆中的酶水解。建立一种高效、快速的短DNA/RNA探针筛选方法，将加快我国生物传感器技术在其他领域的商业化进程。

DESCRIPTION (provided by applicant): We describe an integrated approach for rapid discovery of small, structurally defined nucleic acid "probe" sequences that bind with high affinity and selectivity to a target of interest. Such targets are proteins, bio-terror agents, pathogenic organisms and viruses. The probes will be engineered into proprietary AlloSwitchTM indicators that respond rapidly to the presence of the target, giving rise to a fluorescent or luminescent signal. In this phase I project we propose to rapidly screen and isolate high-affinity combining probes (c-probes). We will use high throughput methods to isolate at least the known probe sequences specific for alpha-thrombin and platelet derived growth factor (PDGF) from a defined library containing billions of DNA sequences. Alternative c-probes for thrombin and PDGF will be sought in libraries that have a predefined secondary structure. In phase II, the approach will be expanded to include a wider variety of structural motifs and new targets. The targets for discovery of probes and creation of AlloSwitches will include proteins involved in clotting and thrombosis, and growth factors involved in wound healing, angiogenesis, and proliferative diseases. AlloSwitch based assays will assist in the discovery of small molecules that can be used for therapies to regulate the interactions of these proteins in blood. This stands in contrast to therapies based on aptamers or antibodies, which are difficult to protect from hydrolysis by enzymes in plasma. Establishing a highly efficient and rapid screening approach for short DNA/RNA probe discovery will accelerate the commercialization of our biosensor technology in other fields.