Rapid isolation of high-affinity DNA aptamers for small-molecule targets via nuclease-assisted SELEX

通过核酸酶辅助 SELEX 快速分离小分子靶标的高亲和力 DNA 适体

• 批准号: 2135005
• 负责人: Yi Xiao
• 金额: $ 35.45万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2135005&HistoricalAwards=false
• 关键词: Rapid; isolation; affinity; DNA; aptamers

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Yi Xiao and her research team in the Department of Chemistry and Biochemistry at Florida International University (FIU) seek to develop a widely usable method for the rapid generation of DNA-based bioreceptors, termed aptamers, which selectively recognize small molecules targets. Aptamers have shown promise as key sensing components for point-of-use, small-molecule biosensing detectors that recognize target molecules by aptamer binding to the targets, making them potentially valuable for scientific, clinical, and forensic applications. However, current methods for aptamer generation are labor intensive, time consuming, and typically yield aptamers that are unable to clearly tell the difference between molecules of similar shape and size. The proposed approach produces high-quality aptamers with superior recognition properties and significantly accelerates the aptamer isolation process. Moreover, this technique might replace existing methods for aptamer generation. In parallel with these research efforts, Professor Xiao, in partnership with Hispanic-serving and historically black colleges and universities, as well as student training programs at FIU, plans a multi-level outreach effort focusing on underrepresented minority (URM) students. These activities will provide students with opportunities to participate in scientific research and encourage them to pursue careers in science, technology, engineering, and mathematics (STEM) fields. In pursuit of this, Dr. Xiao gives science seminars and presentations, organizes summer science camps, and provides summer internships. Leveraging the high structural specificity and efficiency of flap endonuclease 1 (FEN1), Professor Xiao and her research team seek to develop an innovative and generalizable nuclease-assisted aptamer isolation technique, termed NA-SELEX, for the rapid isolation of high-affinity, small-molecule-binding aptamers. To establish this technology, a model SELEX system is first used to systematically study and optimize FEN1-based strand separation. The feasibility and generalizability of NA-SELEX is then demonstrated by isolating high-affinity aptamers for different small-molecule targets with clinical and forensic relevance. Finally, the sensing capabilities of the resulting aptamers is assessed by fabricating electrochemical aptamer-based sensors for analyte detection in biological samples. This new approach has the potential to efficiently generate high-affinity, customizable aptamers on demand for any small-molecule target and greatly facilitate the development of sensing elements for the detection of small molecules for various applications such as food safety, environmental monitoring, therapeutic treatment, and medical diagnostics. In addition, an educational outreach program at FIU increases the exposure of graduate, undergraduate, and high school URM students to STEM fields and promotes an inclusive environment for URMs engaged in STEM.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在化学系化学测量和成像项目的支持下，佛罗里达国际大学 (FIU) 化学与生物化学系的肖毅教授和她的研究团队寻求开发一种广泛使用的方法，用于快速生成基于 DNA 的生物受体（称为适体），它可以选择性地识别小分子靶标。适配体已显示出作为使用点小分子生物传感检测器的关键传感组件的前景，这些检测器通过适配体与目标结合来识别目标分子，这使得它们对科学、临床和法医应用具有潜在价值。然而，目前的适体生成方法是劳动密集型、耗时的，并且通常产生的适体无法清楚地区分相似形状和大小的分子之间的差异。所提出的方法产生具有卓越识别特性的高质量适体，并显着加速了适体分离过程。此外，该技术可能会取代现有的适体生成方法。在开展这些研究工作的同时，肖教授与拉美裔和历史悠久的黑人学院和大学以及佛罗里达国际大学的学生培训项目合作，计划开展多层次的推广工作，重点关注代表性不足的少数族裔 (URM) 学生。这些活动将为学生提供参与科学研究的机会，并鼓励他们在科学、技术、工程和数学（STEM）领域从事职业。为了实现这一目标，肖博士举办科学研讨会和演讲，组织夏季科学营，并提供暑期实习。利用瓣核酸内切酶1（FEN1）的高结构特异性和效率，肖教授和她的研究团队寻求开发一种创新且可推广的核酸酶辅助核酸适体分离技术，称为NA-SELEX，用于快速分离高亲和力、小分子结合的核酸适体。为了建立这项技术，首先使用模型 SELEX 系统来系统地研究和优化基于 FEN1 的链分离。然后通过分离具有临床和法医学相关性的不同小分子靶标的高亲和力适体来证明 NA-SELEX 的可行性和普遍性。最后，通过制造用于生物样品中分析物检测的电化学适体传感器来评估所得适体的传感能力。这种新方法有潜力根据任何小分子靶标的需要高效生成高亲和力、可定制的适体，并极大地促进用于检测小分子的传感元件的开发，用于食品安全、环境监测、治疗和医学诊断等各种应用。此外，FIU 的教育推广计划增加了研究生、本科生和高中 URM 学生对 STEM 领域的接触，并为从事 STEM 的 URM 提供了一个包容性的环境。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。