Simple DNA/RNA Probes for Protein Targets

描述(由申请人提供)：我们描述了一种用于高通量筛选适体TM的集成方法(HTSATM，正在申请专利)，以发现与感兴趣的靶结合具有高亲和力和选择性的小的、结构定义的核酸适体序列。这些目标是蛋白质、生物恐怖因子、病原体和病毒。这些适配子将被设计成我们专利的allSwitchTM指示器的“探头”序列，这些指示器迅速地重新释放到目标的存在，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的HT-适配子是可行的。我们已经使用HTSA分离了先前已知的与人凝血酶(从DNA发夹环)和人凝血因子IXa(从具有串联发夹和内部环的RNA)具有纳摩尔亲和力的适配子，以及一个对己糖具有明显专一性的未被预测的适配子(来自DNA发夹)，其亲和力在已知的小分子适配子的前三分之一。从而论证了HTSA的可行性。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。这与直接基于适配子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，也很难作为药物制成药丸。该项目还将加快我们的生物传感器技术在其他领域的商业化进程。 与公共卫生相关：我们提出了一种快速发现小的、结构定义的核酸“HT-适配子”序列的综合方法，该序列与蛋白质和其他靶标具有高亲和力和选择性。这种方法将被用于制造传感器，以发现小分子作为针对涉及伤口愈合和血栓形成的选定蛋白质靶点的候选药物。除了这个项目外，高亲和力的HT-适配子的发现将被用于检测水传播的病原体、生物战剂和诊断应用，与基于抗体的试剂相比，成本预计将降低10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.