Streamlining HTS Assay Development through Direct Selection of Structure-Switching Aptamers

项目摘要/摘要 高通量筛选(HTS)是发现靶酶新药先导的有效方法。 在高温超导检测中，目标酶的活性通常通过量化小分子或辅因子来评估。 由酶产生或消耗的。虽然抗体是小分子检测的支柱，因为- 说，它们确实有严重的局限性，这在很大程度上与使小分子功能化的挑战有关 在不掩盖关键功能基团的情况下实现目标。结果，目标分子与载体的偶联有利于- 用于产生抗体的Tein是费力的，而且通常会降低产生的抗体的特异性。另外-- 此外，使用小分子结合抗体的HTS检测要求靶标的标记版本 作为化验中的竞争者而生产。我们认为DNA结构转换(SS)生物传感器可以 克服这些限制，因为这些传感器在目标结合时提供直接荧光读数，并且 不需要对目标进行共价标记。此外，这些生物传感器利用核酸AP- 调节剂，可以使用体外选择方法产生，这种方法不一定需要靶标 被修改或固定。虽然SS生物传感器在高温超导和其他小型生物传感器中具有巨大的应用潜力，但它的应用前景十分广阔。 分子检测分析，目前可用于产生这些生物传感器的协议是时间- 耗费精力，有时甚至不可靠。因此，我们提出了一种改进的SS生物菌株的体外筛选方法。 预计将提供更有效的功能序列丰富的传感器。这两项措施都将减少 为新的小分子靶标制造生物传感器所需的时间和提高成功率 利息。在目标1中，我们将开发并实现这种选择方法来生成用于Coen-1的SS生物传感器。 酶A(CoA)。在目标2中，我们将利用这种生物传感器来产生荧光偏振HTS分析 辅酶A是组蛋白乙酰转移酶(HATS)的产物。这项研究的立竿见影的影响将是 经过验证的HTS检测方法用于筛选HATS，这将解决在药物发现方面尚未满足的重大需求 表观遗传性疾病的数量，包括神经疾病、癌症和心血管疾病。从一个 更广阔的前景，这项研究将提供一种快速可靠的方法来产生SS生物传感器 小分子靶标，这将加快高温超导检测各种酶类药物靶标的发展。

Project Summary/Abstract High-throughput screening (HTS) is a powerful method for the discovery of new drug leads for target enzymes. In HTS assays, the activity of the target enzyme is often evaluated by quantifying a small-molecule or cofactor that is produced or consumed by the enzyme. While antibodies are a mainstay of small-molecule detection as- says, they do have severe limitations, which are largely tied the challenge of functionalizing small molecule targets without masking key functional groups. As a result, conjugation of the target molecule to a carrier pro- tein for antibody generation is laborious and often decreases the specificity of the antibodies generated. Addi- tionally, HTS assays that use small-molecule binding antibodies require that a labeled version of the target be produced to act as a competitor in the assay. We propose that DNA structure switching (SS) biosensors can overcome these limitations, as these sensors provide a direct fluorescence readout upon target binding, and do not require that the target be covalently labeled. Additionally, these biosensors utilize nucleic acid ap- tamers, which can be generated using in vitro selection methods that do not necessarily require that the target be modified or immobilized. While SS biosensors hold tremendous potential for use in HTS and other small- molecule detection assays, the currently available protocols for generating these biosensors are time- consuming and at times unreliable. Thus, we propose an improved method for the in vitro selection of SS bio- sensors that is anticipated to provide more efficient enrichment of functional sequences. This will both reduce the time required and increase the success rate for generating biosensors to new small-molecule targets of interest. In Aim 1, we will develop and implement this selection method to generate a SS biosensor for Coen- zyme A (CoA). In Aim 2, we will utilize this biosensor to produce a fluorescence polarization HTS assay for CoA, the product of histone acetyltransferases (HATs). The immediate impact of this research will be an im- proved HTS assay for screening HATs, which will address a significant unmet need in drug discovery for a number of epigenetic diseases including neurological disorders, cancers, and cardiovascular disease. From a broader perspective, this research will provide a rapid and reliable method for generating SS biosensors for small-molecule targets, which will accelerate development of HTS assays for diverse enzyme drug targets.