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.

概括 适体（基于寡核苷酸的受体）是潜在的通用受体，可实现精确、准确、 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体最近分离出多个优秀的适配体 目前正在集成到下一代家庭、实时和临床实验室测试中 （自动化、并行）分析。例如，在哥伦比亚大学医学中心 (CUMC)，我们位于 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 在临床决策分析中的角色的背景下对实际患者样本进行验证的过程 - 制作。此外，我们的合作者正在与我们一起测试第四代可卡因适体。 在成瘾动物模型中发表了血清素适体。 尽管取得了这些进步，并且我们已经进行了 100 多次成功的选择，但仍有一些重要的个人 目标甚至整个类别的目标，为此多次尝试以最小的量分离适体 亲和力，意外失败。我们现在介绍我们从这些失败中学到的教训，并描述一个系统的 即使是最具挑战性的目标，也可以分离出临床上有用的适体。在这个过程中，我们 引入概念上的新选择，包括“等排-”、“模拟-”和“诱饵-开关”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以应对挑战 分析物。我们将追求：（1）复杂的目标，其溶解度低于捕获低亲和力所需的溶解度 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 从小型寡核苷酸文库中分离出来（丙戊酸）； (3) 需要的免疫抑制剂 大的或延伸的和浅的结合袋（环孢素和西罗莫司，其类似物依维莫司，所有 疏水性、大且表位较差）。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，并像我们一样进行验证 准备实验室开发的测试 (LDT) 并提交给纽约州卫生部。 除了高价值目标的特定受体之外，这项资助的重要成果不会是个体快速 程序，因为它是该领域的标准，但是协议的多步骤实施的算法。这 通过解决广泛认可的问题，在标准实践上大幅增加努力是完全合理的。 临床化学：能够对无法进行足够快速分析的分析物进行定量 临床决策的准确性和精确性以及频繁的“需求点”监测。