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.

摘要 适配子(基于寡核苷酸的受体)是潜在的通用Go-to受体， 然而，在临床化学中，小分子的快速测定。方法的系统改进 开发高质量的小分子适配子最近导致分离出多个优秀的适配子 目前正在集成到家庭、实时和临床实验室的下一代测试中 (自动化、并行)分析。例如，在哥伦比亚大学医学中心(CUMC)，我们处于 与苯丙氨酸、万古霉素、妥布霉素和尿素的原始适配子的关键连接，都在 根据实际患者样本在临床决策分析中的作用对其进行验证的过程- 制作。此外，我们的合作者正在测试第四代可卡因适体，与我们的 公布了成瘾动物模型中的5-羟色胺适配子。 尽管取得了这些进步，在我们身后有100多个成功的选择，但还是有一些重要的个人 靶标，甚至整个类别的靶标，对于这些靶标，多次尝试分离适配子，即使是最少的 亲和力，竟然失败了。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 为即使是最具挑战性的靶点分离临床上有用的适配子的方法。在这个过程中，我们 在概念上引入新的选择，包括‘isstere-’、‘模拟-’和‘诱饵和切换’选择。。 通过三个目标，我们将开发算法来选择临床上有用的适体来挑战 分析物。我们将追求：(1)溶解度低于捕捉低亲和力所需的复杂靶标 适配子(伏立康唑和25-羟基维生素D3)；(2)与适配子结合能低的阴离子靶标 从小的寡核苷酸文库分离(丙戊酸)；和(3)需要以下两种之一的免疫抑制剂 大的或延长的浅绑扎袋(环孢素A和西罗莫司，其类似物伊维莫司，所有 疏水性、大和表位差)。新分离的适配子将不断优化，以达到 临床测试所需的亲和力，转换为适当的传感器格式，并进行验证，就像我们 准备实验室开发的测试(LDT)，以提交给纽约卫生部。 除了高价值靶标的特定受体外，这项拨款的重要结果不会是个别迅速的。 程序，因为它一直是该领域的标准，但多步骤执行协议的算法。这个 在标准实践上大幅增加的努力是完全合理的，因为解决了 临床化学：能够对没有快速分析的分析物进行量化 确保临床决策的准确性和精确性，并经常在“需要时”进行监测。