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

在化学系化学测量和成像计划的支持下，佛罗里达国际大学(FIU)化学和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适用的方法来快速生成基于DNA的生物受体，称为适体，它选择性地识别小分子靶标。适配子已显示出作为关键传感组件的前景，用于通过适配子与靶结合识别靶分子的使用点、小分子生物传感检测器，使其在科学、临床和法医应用中具有潜在的价值。然而，目前产生适体的方法劳动强度大、耗时长，而且通常产生的适体不能清楚地区分相似形状和大小的分子之间的区别。所提出的方法产生了高质量的适体，具有良好的识别性能，并显着加快了适体分离过程。此外，这项技术可能会取代现有的适配子生成方法。在这些研究工作的同时，肖教授与服务于西班牙裔和历史上一直是黑人的学院和大学以及FIU的学生培训项目合作，计划开展一项多层次的外展活动，重点关注代表人数不足的少数族裔(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)领域追求职业生涯。为了追求这一目标，肖博士举办了科学研讨会和演讲，组织了夏季科学夏令营，并提供了暑期实习机会。利用FEN1的高结构特异性和高效率，肖教授和她的研究团队试图开发一种创新的、可推广的核酸酶辅助适配子分离技术，称为NA-SELEX，用于快速分离高亲和力、小分子结合的适配子。为了建立这项技术，首先使用一个模型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配子，证明了NA-SELEX的可行性和普适性。最后，通过制作基于电化学适配子的传感器来检测生物样品中的分析物，评估了所产生的适配子的传感能力。这种新方法有可能根据任何小分子靶标的需求高效地产生高亲和力、可定制的适配子，并极大地促进用于检测小分子的传感元件的开发，这些应用包括食品安全、环境监测、治疗治疗和医疗诊断。此外，FIU的一项教育推广计划增加了研究生、本科生和高中URM学生接触STEM领域的机会，并促进了从事STEM的URM的包容性环境。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.