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

通过直接选择结构转换适体简化 HTS 检测开发

• 批准号: 9202010
• 负责人: Jennifer Margaret Heemstra
• 金额: $ 23.49万
• 依托单位: BELLBROOK LABS, LLC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-13 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9202010
• 关键词: Address; Affinity; Antibodies; Antibody Formation; Antibody Specificity; Asses; Binding; Biological Assay; Biosensor; Cardiovascular Diseases; Carrier Proteins; Chemical Structure; Cleaved cell; Coenzyme A; Coenzymes; Coupled; DNA; DNA Structure; Detection; Development; Disease; Dose; Drug Targeting; Engineering; Enzymes; Epigenetic Process; Event; Family; Fluorescence; Fluorescence Polarization; Generations; High-Throughput Nucleotide Sequencing; Immobilization; In Vitro; Investigation; Label; Libraries; Malignant Neoplasms; Masks; Methodology; Methods; Methyltransferase; Mole the mammal; Niacinamide; Nucleic Acids; Nucleotides; Phase; Phosphotransferases; Play; Positioning Attribute; Process; Proteins; Protocols documentation; Reaction; Reagent; Research; Sensitivity and Specificity; Signal Transduction; Stream; Streptavidin; Structure; Sulfhydryl Compounds; Time; Tweens; Universities; Utah; Variant; abstracting; aptamer; assay development; base; cofactor; drug discovery; fluorophore; functional group; high throughput screening; histone acetyltransferase; improved; in vivo; interest; method development; nervous system disorder; novel therapeutics; nuclease; prevent; screening; sensor; small molecule; success; therapeutic target

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.

项目摘要/摘要 高通量筛选（HTS）是发现靶酶新药物铅的强大方法。 在HTS测定中，通常通过量化小分子或辅因子来评估靶酶的活性 这是由酶产生或消耗的。而抗体是小分子检测的中流 说，它们确实有严重的局限性，这在很大程度上束缚了官能化小分子的挑战 目标没有掩盖关键官能团。结果，靶分子与载体前轭的结合 用于抗体产生的TEIN是费力的，通常会降低产生的抗体的特异性。添加 使用小分子结合抗体的HTS测定法需要标记的目标版本 生产是作为测定法的竞争对手。我们建议DNA结构切换（SS）生物传感器可以 克服这些局限性，因为这些传感器在目标结合时提供了直接的荧光读数，并且 不需要目标价值标记。此外，这些生物传感器还利用了核酸ap- 驯服者，可以使用体外选择方法生成，而这些方法不一定要求目标 被修改或固定。虽然SS生物传感器具有在HTS和其他小型中使用的巨大潜力 分子检测测定法，当前可用的生物传感器的方案是时间 - 消费和有时不可靠。因此，我们提出了一种改进的方法，用于体外选择SS Bio- 预计将提供更有效的功能序列的传感器。这两个都会减少 所需的时间并提高生物传感器到新的小分子目标的成功率 兴趣。在AIM 1中，我们将开发和实施此选择方法，以生成用于COEN-的SS生物传感器 Zyme A（COA）。在AIM 2中，我们将利用该生物传感器生成荧光极化HTS分析 COA，组蛋白乙酰转移酶（HATS）的产物。这项研究的直接影响将是一个重要的 证明了用于筛选帽子的HTS测定法，这将解决药物发现的重要需求 包括神经系统疾病，癌症和心血管疾病在内的表观遗传疾病的数量。来自 更广泛的观点，这项研究将为生成SS生物传感器的快速可靠方法 小分子靶标，该靶标将加速HTS测定的多种酶药物靶标。