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

基本信息

批准号：
10647742
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
项目类别：
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/10647742
关键词：
Academic Medical Centers Address Affinity Alanine Algorithms Animal Model Binding Biological Assay Breakthrough device Calcifediol Charge Cholecalciferol Clinical Clinical Chemistry Cocaine Complex Cyclosporine Decision Making Dominant-Negative Mutation Drug Monitoring Elements Epitopes Failure Free Energy Generations Grant Health Home Hydrophobic Surfaces Hydrophobicity Immunosuppressive Agents Individual Laboratories Learning 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 biomarker discovery clinical application clinical decision-making design functional group improved insight next generation point of care prevent programs rapid test receptor research clinical testing sensor small molecule valproate

项目摘要

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 APATMER（基于寡核苷酸的受体）可能是一般的通用受体，具有精度和准确的受体，然而快速，测定临床化学中小分子。系统改进方法为小分子开发高质量的适体导致隔离多个出色的适体目前正在集成在国内，实时和临床实验室的下一代测试中（自动化，平行）分析。例如，在哥伦比亚大学医学中心（CUMC），我们在与苯丙氨酸，万古霉素，毒素和尿素的原始适体的关键交界处，都在在临床决策中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5-羟色胺适体。尽管有这些进步，并且在我们身后有100多个成功的选择，但有重要的个人目标，甚至整个目标，为此，多次尝试将适体分离为最小亲和力，意外失败。现在，我们介绍我们从这些失败中学到的知识，并描述了系统的即使是最具挑战性的目标，隔离临床上有用的适体的方法。在此过程中，我们从概念上介绍新的选择，包括“ Isostere-”，“ Analog-”和“ Bait and-Switch”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来挑战分析物。我们将追求：（1）溶解性低于捕获低亲和力所需的复杂靶标适体（Voriconazole和25-OH维生素D3）；（2）与适体的结合自由能低的阴离子靶标从小寡核苷酸库（丙戊酸）中分离出来；（3）需要的免疫抑制剂大型或延伸且浅的结合口袋（环孢菌素和西罗莫司（Sirolimus）疏水，大，表位较差）。新隔离的适体将不断优化以达到临床测试所需的亲和力，变成适当的传感器格式，并验证了我们准备实验室开发的测试（LDT），以提交给纽约州卫生部。除了高价值目标的特定接收器之外，这笔赠款的重要结果将不是个人快速的该过程是该领域的标准配置，但是用于多步实施协议的算法。通过解决广泛认识的问题，超出标准实践的努力大幅提高是合理的临床化学：实现没有足够快速测定的分析物的定量临床决策的准确性和精度，并且经常在“需要”监测。