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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要高通量筛选 (HTS) 是发现靶酶新药先导物的强大方法。在 HTS 测定中，通常通过量化小分子或辅因子来评估目标酶的活性由酶产生或消耗的物质。虽然抗体是小分子检测的支柱—— 说，它们确实有严重的局限性，这在很大程度上与小分子功能化的挑战有关目标而不掩盖关键功能组。结果，目标分子与载体亲的缀合用于抗体生成的 tein 是费力的，并且通常会降低生成的抗体的特异性。阿迪- 理论上，使用小分子结合抗体的 HTS 测定需要标记的靶标版本生产作为测定中的竞争者。我们建议 DNA 结构转换（SS）生物传感器可以克服了这些限制，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- 驯服者，可以使用体外选择方法产生，不一定要求目标被修改或固定。虽然 SS 生物传感器在 HTS 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，目前用于生成这些生物传感器的可用协议是时间- 消耗且有时不可靠。因此，我们提出了一种改进的 SS 生物体外选择方法。预计将提供更有效的功能序列丰富的传感器。这都会减少所需的时间并提高生成生物传感器以检测新的小分子目标的成功率兴趣。在目标 1 中，我们将开发并实施这种选择方法，以生成用于 Coen- 的 SS 生物传感器。酶 A (CoA)。在目标 2 中，我们将利用该生物传感器对以下物质进行荧光偏振 HTS 测定： CoA，组蛋白乙酰转移酶 (HAT) 的产物。这项研究的直接影响将是经过验证的 HTS 检测可用于筛选 HAT，这将解决药物发现中未满足的重大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。来自一个从更广阔的角度来看，这项研究将为生成SS生物传感器提供一种快速可靠的方法小分子靶标，这将加速针对不同酶药物靶标的 HTS 检测的开发。