Inorganic-aptamer hybrids for live cell imaging

用于活细胞成像的无机适体杂交体

• 批准号: 2002956
• 负责人: Edith Glazer
• 金额: $ 37.2万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2002956&HistoricalAwards=false
• 关键词: Inorganic; aptamer; hybrids; live; cell

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Edith Glazer from the University of Kentucky to develop a short RNA sequence, called an aptamer, that can be observed to go through its life cycle in a cell using fluorescence microscopy. RNA has the task of translating the genetic information that is stored in DNA to create the proteins that serve as the workhorses in the cells of all living organisms. By observing how the amount of RNA changes in a cell, scientists can study how this flow of information is controlled in a time-dependent manner. The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method is used to evolve RNA aptamers that bind to a small molecule containing a ruthenium (Ru) metal ion. This inorganic compound is designed to light up when it binds to form a complex with its specific RNA partner, as the two fit together in lock and key fashion. The light emitted by the complex is visualized by fluorescence microscopy and other optical methods to “see” the RNA aptamer as it is being produced and later degraded. The research project provides training to undergraduate and graduate students in organic and inorganic synthesis, molecular biology, and various light microscopy techniques. The project will also be integrated into an educational program aimed at increasing retention and success rates in chemistry for students that are women and members of underrepresented groups.The ability to accurately track and quantify RNA levels or localization, either on the subcellular or tissue levels, is important to the understanding of the role of RNA in the regulation of biological processes. This research project develops new emissive ruthenium (Ru(II)) compounds for tracking RNA in live cells through the creation of optimized pairs of luminescent compounds and RNA aptamers. The Ru(II) compounds do not suffer from photobleaching or excited state conformational changes, overcoming a key obstacle in the field of optical RNA reporters. The research increases the understanding of how synthetic modification alters excited state energies and relaxation pathways in luminescent Ru(II) complexes, and how specific modifications determine RNA selectivity. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a powerful method to develop nucleic acids that bind target small molecules, and the combination of SELEX with modular chemical modification of the Ru(II) complex provides optimized binding partners. This project combines the techniques of synthetic chemistry, molecular biology, and microscopy to develop new tools to improve the understanding of RNA biology.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.

有了这个奖项，化学部的生命过程化学计划正在资助肯塔基州大学的Edith Glazer博士开发一种称为适体的短RNA序列，可以使用荧光显微镜观察到它在细胞中的生命周期。RNA的任务是翻译储存在DNA中的遗传信息，以创造蛋白质，这些蛋白质是所有生物体细胞中的主力。通过观察细胞中RNA的量如何变化，科学家们可以研究这种信息流是如何以时间依赖的方式控制的。 通过指数富集的配体系统进化（SELEX）方法用于进化与含有钌（Ru）金属离子的小分子结合的RNA适体。这种无机化合物被设计成当它与其特定的RNA伴侣结合形成复合物时发光，因为两者以锁和钥匙的方式结合在一起。 复合物发出的光通过荧光显微镜和其他光学方法可视化，以“看到”RNA适体，因为它正在产生和随后降解。该研究项目为本科生和研究生提供有机和无机合成，分子生物学和各种光学显微镜技术的培训。该项目还将被纳入一个教育计划，旨在提高学生的保留率和化学的成功率是妇女和代表性不足的群体的成员。准确跟踪和定量RNA水平或定位的能力，无论是在亚细胞或组织水平，是重要的RNA在生物过程的调节中的作用的理解。该研究项目开发了新的发射性钌（Ru（II））化合物，通过创建优化的发光化合物和RNA适体对来跟踪活细胞中的RNA。Ru（II）化合物不受光漂白或激发态构象变化的影响，克服了光学RNA报告领域的一个关键障碍。该研究增加了对合成修饰如何改变发光Ru（II）络合物中的激发态能量和弛豫途径以及特定修饰如何决定RNA选择性的理解。通过指数富集的配体系统进化（SELEX）是开发结合靶小分子的核酸的有力方法，并且SELEX与Ru（II）络合物的模块化化学修饰的组合提供了优化的结合配偶体。该项目结合了合成化学、分子生物学和显微镜技术，开发了新的工具，以提高对RNA生物学的理解。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。