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: 2333820
• 负责人: Edith Glazer
• 金额: $ 37.2万
• 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: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2333820&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，化学部门的生命过程化学项目资助肯塔基大学的Edith Glazer博士开发一种短RNA序列，称为适体，可以使用荧光显微镜观察细胞中的生命周期。RNA的任务是翻译存储在DNA中的遗传信息，以创造蛋白质，这些蛋白质在所有生物体的细胞中充当主力。通过观察细胞中RNA的数量是如何变化的，科学家们可以研究这种信息流是如何以一种依赖时间的方式被控制的。配体的系统进化指数富集（SELEX）方法用于进化RNA适体结合到一个小分子含有钌（Ru）金属离子。这种无机化合物被设计成在与特定的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。通过荧光显微镜和其他光学方法可以看到复合物发出的光，以“看到”RNA适体的产生和随后的降解过程。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方面的培训。该项目还将被纳入一项教育计划，旨在提高女性和代表性不足群体学生的化学保留率和成功率。准确跟踪和量化RNA水平或定位的能力，无论是在亚细胞水平还是组织水平，对于理解RNA在生物过程调节中的作用是很重要的。本研究项目通过创建优化的发光化合物和RNA适配体对，开发新的发光钌（Ru(II)）化合物，用于跟踪活细胞中的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报告器领域的关键障碍。该研究增加了对合成修饰如何改变发光Ru（II）配合物的激发态能量和弛豫途径，以及特异性修饰如何决定RNA选择性的理解。SELEX （Systematic Evolution of Ligands by Exponential Enrichment）是开发结合目标小分子的核酸的有力方法，SELEX与Ru（II）配合物的模块化化学修饰相结合提供了优化的结合伙伴。该项目结合了合成化学、分子生物学和显微镜技术来开发新的工具，以提高对RNA生物学的理解。该奖项反映了美国国家科学基金会的法定使命，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。