Simple DNA/RNA Probes for Protein Targets

描述（由申请人提供）：我们描述了一种用于适体TM的高通量筛选（HTSATM，专利申请中）的综合方法，以发现小的、结构上确定的核酸适体序列，其以高亲和力和选择性结合到感兴趣的靶标。这些目标是蛋白质、生物恐怖剂、病原体和病毒。这些适体将被设计到我们的专利AlloSwitchTM指示剂的“探针”序列中，该指示剂可快速响应目标的存在，产生荧光或发光信号。在第一阶段，我们证明了快速筛选和分离高亲和力HT-适体是可行的。我们已经使用HTSA分离了先前已知的对人凝血酶（来自DNA发夹环）和人凝血因子IXa（来自具有级联发夹和内环的RNA）具有纳摩尔亲和力的适体，以及对己糖具有明显特异性的未预测的适体（来自DNA发夹），其对小分子的亲和力在已知适体的前三分之一中。因此，HTSA的可行性得到了论证。在第二阶段，将扩大这一方法，以包括更广泛的结构基元和新的目标。发现探针和创建AlloSwitch的目标将包括参与凝血和血栓形成的蛋白质，以及参与伤口愈合、血管生成和增殖性疾病的生长因子。基于AlloSwitch的分析将有助于发现可用于治疗的小分子，以调节血液中这些蛋白质的相互作用。这与直接基于适体或抗体的疗法形成对比，适体或抗体难以防止血浆中的酶水解，并且难以配制成药丸中的药物。该项目还将加速我们的生物传感器技术在其他领域的商业化。 公共卫生相关性：我们提出了一种用于快速发现小的、结构上确定的核酸“HT-适体”序列的集成方法，所述核酸“HT-适体”序列以高亲和力和选择性与蛋白质和其他靶标结合。该方法将用于创建传感器，以发现小分子作为药物的候选者，针对参与伤口愈合和血栓形成的选定蛋白质靶点。除了这个项目之外，高亲和力HT-适体的发现将用于检测水性病原体、生物战剂和诊断应用，与基于抗体的试剂相比，预计成本将降低十倍或更多。

DESCRIPTION (provided by applicant): We describe an integrated approach for High Throughput Screening of AptamersTM (HTSATM, patent pending) to discover small, structurally defined nucleic acid aptamer sequences that bind with high affinity and selectivity to a target of interest. Such targets are proteins, bio-terror agents, pathogenic organisms and viruses. These aptamers will be engineered into the "probe" sequence of our patented AlloSwitchTM indicators that re- spond rapidly to the presence of the target, giving rise to a fluorescent or luminescent signal. In phase I we demonstrated that it is feasible to rapidly screen and isolate high-affinity HT-aptamers. We have used HTSA to isolate previously known aptamers with nanomolar affinity for human athrombin (from DNA hairpin loops) and for human coagulation factor IXa (from RNA with concatenated hairpin and internal loops), and an unprece- dented aptamer with apparent specificity for hexose sugars (from DNA hairpins) with affinity in the top third of known aptamers for small molecules. Thus, the feasibility of HTSA has been demonstrated. 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 directly on aptamers or antibodies, which are difficult to protect from hydrolysis by enzymes in plasma and to formulate as pharmaceuticals in pills. This project will also accelerate the commercialization of our biosensor technology in other fields. PUBLIC HEALTH RELEVANCE: We present an integrated approach for rapid discovery of small, structurally defined nucleic acid "HT- aptamer" sequences that bind with high affinity and selectivity to proteins and other targets. The approach will be used to create sensors to discover small molecules as candidates for drugs against selected protein targets involved in wound healing and thrombosis. Beyond this project, discovery of high affinity HT-aptamers will be used in detecting waterborne pathogens, biological warfare agents, and diagnostic applications with costs ex- pected to be reduced by a factor of ten or more compared to antibody-based reagents.