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.

佛罗里达国际大学（FIU）化学与生物化学系的Yi Xiao教授和她的研究团队在化学系化学测量和成像项目的支持下，寻求开发一种广泛可用的方法，用于快速生成基于DNA的生物受体，称为适体，其选择性识别小分子靶标。适体已经显示出作为使用点的关键传感组件的前景，小分子生物传感检测器通过适体结合到目标来识别目标分子，使它们对于科学、临床和法医应用具有潜在价值。然而，目前用于适体产生的方法是劳动密集型的、耗时的，并且通常产生的适体不能清楚地分辨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上级识别特性的高质量适体，并显著加速适体分离过程。此外，这种技术可能取代现有的适体生成方法。在开展这些研究工作的同时，肖教授与西班牙裔和历史上的黑人学院和大学以及佛罗里达大学的学生培训项目合作，计划开展多层次的外展工作，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，并鼓励他们从事科学，技术，工程和数学（STEM）领域的职业。为了追求这一目标，肖博士举办科学研讨会和演讲，组织暑期科学夏令营，并提供暑期实习机会。利用瓣状核酸内切酶1（FEN 1）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的和可推广的核酸酶辅助适体分离技术，称为NA-SELEX，用于快速分离高亲和力，小分子结合适体。为了建立该技术，首先使用模型SELEX系统来系统地研究和优化基于FEN 1的链分离。然后通过分离具有临床和法医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基于电化学适体的传感器来评估所得适体的传感能力。这种新方法有可能有效地产生高亲和力，可定制的适体对任何小分子目标的需求，并大大促进传感元件的发展，用于检测小分子的各种应用，如食品安全，环境监测，治疗性治疗和医疗诊断。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提供包容性环境。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。