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.

项目总结/摘要 高通量筛选（HTS）是发现靶酶的新药先导物的有力方法。 在HTS测定中，通常通过定量小分子或辅因子来评估靶酶的活性 由酶产生或消耗。虽然抗体是小分子检测的支柱， 他们确实有严重的局限性，这在很大程度上与功能化小分子的挑战有关。 目标，而不掩盖关键功能组。因此，靶分子与载体的缀合有利于靶分子的表达。 用于抗体产生的蛋白质是费力的，并且经常降低所产生抗体的特异性。加- 在理论上，使用小分子结合抗体的HTS测定要求靶的标记形式被 在试验中作为竞争者。我们提出DNA结构转换（SS）生物传感器可以 克服这些限制，因为这些传感器在靶结合时提供直接荧光读数，并且 不要求靶被共价标记。此外，这些生物传感器利用核酸AP- 可以使用体外选择方法产生的嵌合体，所述方法不一定要求靶 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， 分子检测分析，目前可用于产生这些生物传感器的方案是时间- 消耗并且有时不可靠。因此，我们提出了一种改进的方法，在体外选择SS生物- 这些传感器预期提供更有效的功能序列富集。这将减少 所需的时间，并提高成功率，为新的小分子目标的生物传感器， 兴趣在目标1中，我们将开发和实施这种选择方法，以生成用于Coen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产生荧光偏振HTS测定， CoA是组蛋白乙酰转移酶（HAT）的产物。这项研究的直接影响将是一个IM- 证明HTS检测筛选HAT，这将解决药物发现的重大未满足的需求， 许多表观遗传疾病，包括神经系统疾病、癌症和心血管疾病。从 从更广泛的角度来看，这项研究将提供一个快速，可靠的方法，为生产SS生物传感器， 小分子靶点，这将加速开发用于不同酶药物靶点的HTS测定。