Analog-based Approaches to Isolation of Aptamers for Challenging Targets

基于模拟的方法来分离具有挑战性的靶标的适体

• 批准号: 10429939
• 负责人: Milan N Stojanovic
• 金额: $ 57.2万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10429939
• 关键词: Academic Medical Centers; Address; Affinity; Alanine; Algorithms; Animal Model; Binding; Biological Assay; Biological Markers; Calcifediol; Charge; Cholecalciferol; Clinical; Clinical Chemistry; Cocaine; Complex; Cyclosporine; Devices; Dominant-Negative Mutation; Drug Monitoring; Elements; Epitopes; Failure; Free Energy; Generations; Grant; Health; Home; Hydrophobic Surfaces; Hydrophobicity; Immunosuppressive Agents; Individual; Laboratories; Libraries; Measurement; Measures; Medical; Methods; Molecular; Monitor; Nucleic Acids; Oligonucleotides; Outcome; Patients; Pharmaceutical Chemistry; Phenylalanine; Procedures; Process; Protocols documentation; Publishing; Role; SDZ RAD; Sampling; Scheme; Serotonin; Shapes; Sirolimus; Solubility; Specificity; Structure; Tacrolimus; Testing; Therapeutic; Time; Tobramycin; Urea; Validation; Valproic Acid; Vancomycin; Voriconazole; addiction; analog; aptamer; base; clinical application; clinical decision-making; design; functional group; insight; next generation; point of care; prevent; programs; rapid test; receptor; research clinical testing; sensor; small molecule; valproate

Summary Aptamers (oligonucleotide-based receptors) are potentially general go-to receptors for precise and accurate, yet rapid, determination of small molecules in clinical chemistry. The systematic improvement of methods to develop high-quality aptamers for small molecules recently led to isolation of multiple outstanding aptamers that are currently being integrated in the next generation of tests for at home, real-time, and clinical laboratory (automatized, parallel) analysis. For example, at Columbia University Medical Center (CUMC), we are at a critical junction with original aptamers for phenyl alanine, vancomycin, tobramycin, and urea, all being in the process of validation on actual patients’ samples in the context of their role in assays for clinical decision- making. Further, our collaborators are testing fourth generation cocaine aptamers in conjunction with our published serotonin aptamers in animal models of addiction. Despite these advances, and with over 100 successful selections behind us, there were important individual targets and even whole classes of targets, for which multiple attempts to isolate aptamers with even minimal affinity, failed unexpectedly. We now present what we learned from these failures and describe a systematic approach to isolate clinically useful aptamers for even the most challenging targets. In the process, we introduce conceptually new selections, including ‘isostere-’, ‘analog-’, and ‘bait-and-switch’ selections. . Through three Aims, we will develop algorithms for selection of clinically useful aptamers to challenging analytes. We will pursue: (1) Complex targets with solubility below that needed to capture even low-affinity aptamers (voriconazole and 25-OH vitamin D3); (2) Anionic targets with low binding free energy to aptamers isolated from small oligonucleotide libraries (valproic acid); and (3) Immunosuppressants that require either large, or extended and shallow binding pockets (cyclosporine and sirolimus, its analog everolimus, all hydrophobic, large, and with poor epitopes). Newly isolated aptamers will be continuously optimized to reach affinities required for clinical testing, turned into appropriate sensor formats, and validated as if we are preparing a laboratory-developed test (LDT) for submission to the NYS Department of Health. Beyond specific receptors for high-value targets, the important outcome of this grant will not be individual rapid procedures as it had been standard in the field, but algorithms for multistep implementation of protocols. The substantial increase in effort over standard practice is fully justified by solving widely recognized problems in clinical chemistry: enabling quantification of analytes for which there are no rapid assays with sufficient accuracy and precision for clinical decision making and frequent at ‘point-of-need’ monitoring.

总结 适体（基于阿替西肽的受体）是用于精确和准确的潜在通用受体， 但在临床化学中快速测定小分子。系统地改进方法， 开发高质量的小分子适体最近导致了多个优秀适体的分离 目前正在集成到下一代测试中，用于家庭，实时和临床实验室 （自动化，并行）分析。例如，在哥伦比亚大学医学中心（Columbia University Medical Center，简称CDC）， 与苯丙氨酸、万古霉素、妥布霉素和尿素的原始适体的关键连接，所有这些都在 在实际患者样本在临床决策分析中的作用背景下，对实际患者样本进行验证的过程- 制作。此外，我们的合作者正在测试第四代可卡因适体， 发表了5-羟色胺适体在成瘾动物模型中的应用。 尽管取得了这些进步，并且在我们身后有100多个成功的选择，但仍然有重要的个人 目标，甚至整个类别的目标，其中多次尝试分离适体，即使是最小的 亲和力，意外失败。我们现在介绍我们从这些失败中学到的东西，并描述一个系统的 这是一种分离临床上有用的适体的方法，即使是最具挑战性的靶标。在这个过程中我们 引入概念上的新选择，包括“等排体”、“模拟”和“诱饵和开关”选择。. 通过三个目标，我们将开发用于选择临床上有用的适体的算法，以挑战 分析物。我们将追求：（1）溶解度低于捕获甚至低亲和力所需的复杂靶标 适体（伏立康唑和25-OH维生素D3）;（2）与适体结合自由能低的阴离子靶标 从小的寡核苷酸文库中分离的（丙戊酸）;和（3）需要 大的、或延伸的、浅的结合口袋（环孢菌素和西罗莫司、其类似物依维莫司，所有 疏水的、大的和具有差的表位）。新分离的适体将不断优化，以达到 临床测试所需的亲和力，转化为适当的传感器格式，并进行验证， 准备一个实验室开发的测试（LDT）提交给纽约州卫生部。 除了高价值目标的特定受体之外，这项赠款的重要成果将不会是个人的快速发展。 程序，因为它已经在该领域的标准，但算法的多步骤实施的协议。的 在标准实践基础上大量增加工作量是完全合理的，因为它解决了广泛认识到的问题， 临床化学：能够定量分析物，对于这些分析物，没有足够的 临床决策的准确性和精确性，以及在“需要时”的频繁监测。