Manipulating nucleic acids: applications in RNA biosensing, single-molecule analysis, and DNA nanotechnology

项目摘要 我们的愿景是开发，应用和推进新技术在广泛的主题程序的操纵 的核酸。这项研究主要集中在基于RNA的应用上，这既是驱动的，也是增强的 我们在RNA研究所独特的环境，使容易合作和思想的交叉授粉， 跨领域的技术。RNA和RNA技术的重要性从未如此清晰， RNA病毒（SARS-CoV-2）由RNA生物传感工具和基于mRNA的疫苗管理。现在好了 认为RNA在生物学中扮演着复杂的角色，远远超出了“中心法则”。阐明了许多 RNA的功能和生物医学应用需要对技术开发进行投资。在这一提议中， 在接下来的五年里，我们的目标是在三个主要领域推进技术：1）DNA纳米开关 作为报告分子相互作用的可编程纳米级试剂，2）微力显微镜 (CFM)用于高通量单分子探测和分析，以及3）DNA纳米技术用于以下应用： 药物输送和传感。每个领域都建立在我们的优势和过去的表现之上。 在第一个领域，我们使用DNA纳米开关作为“智能”试剂，在结合目标时改变构象 分子。在过去成功的基础上，我们将扩大这种创新方法，使低成本和简单的RNA 用于各种RNA的检测、定量和纯化。这些包括检测RNA修饰， 调节lncRNA的检测，以及RNA和RNA复合物的纯化。在第二个领域，我们将 在RNA和DNA相互作用分析中开发和执行微力显微镜（CFM）的分析 和RNA结构-功能探测。特别是，我们将研究弱的基本热力学。 研究了DNA和RNA的相互作用，开发了一种新的RNA单分子结构探测方法，并研究了 临床前治疗性核酶的结构-功能关系。在第三个领域，对我们来说相对较新， 实验室，我们扩大工作，分析和开发DNA纳米技术的应用，包括药物输送。 具体来说，我们感兴趣的是理解和“编程”DNA纳米结构的生物稳定性， 协调化学连接和触发从DNA纳米结构释放分子有效载荷。 该提案旨在实施雄心勃勃的研究和开发计划， 几个生物医学项目。这些发展的影响将超越个别项目，因为我们的 核心技术和方法越来越多地被其他群体采用。在第一个MIRA术语中，我们已经证明 我们在多个领域开发有影响力的技术和推进创新科学的能力。在第二个任期内， 我们的目标是显示我们的研究工作的可持续性，巩固我们的利基，保持高生产力， 最终产生有益于科学界的知识和工具。

Project Summary Our vision is to develop, apply, and advance new technologies in the broad thematic program of the manipulation of nucleic acids. The research largely focuses on RNA-based applications, which is both driven and enhanced by our unique environment at The RNA Institute, enabling easy collaboration and cross pollination of ideas and techniques across fields. The importance of RNA and RNA technologies has never been clearer, with a pandemic RNA virus (SARS-CoV-2) being managed by RNA biosensing tools and mRNA-based vaccines. It is now well accepted that RNA plays a complex role in biology that goes far beyond the “central dogma”. Elucidating many RNA functions and biomedical applications requires investment in technology development. In this proposal, carried out over the next five years, we aim to advance technology in three main areas: 1) DNA nanoswitches as programmable nanoscale reagents for reporting molecular interactions, 2) the Centrifuge Force Microscope (CFM) for high throughput single molecule probing and analysis, and 3) DNA nanotechnology for applications in drug delivery and sensing. Each of these areas builds on our strengths and past performance. In the first area, we use DNA nanoswitches as “smart” reagents that change conformation upon binding a target molecule. Building on past success, we will expand this innovative method to enable low cost and simple RNA detection, quantification, and purification for various RNAs. These include detection of RNA modifications, detection of regulatory lncRNAs, and purification of RNA and RNA complexes. In the second area, we will develop and execute assays for the Centrifuge Force Microscope (CFM) in RNA and DNA interaction analysis and RNA structure-function probing. In particular, we will investigate fundamental thermodynamics of weak interactions in DNA and RNA, develop a new method for single-molecule structural probing of RNA, and study the structure-function relationship of a pre-clinical therapeutic ribozyme. In the third area, relatively new for our lab, we expand work to analyze and develop DNA nanotechnology for applications including drug delivery. Specifically, we are interested in understanding and “programming” biostability into DNA nanostructures, and in coordinating chemical attachment and triggered release of molecular payloads from DNA nanostructures. This proposal aims to carry out ambitious research and development plans performed within the context of several biomedical projects. The impact of the developments will extend beyond the individual projects, as our core technologies and methods are increasingly adopted by other groups. In the first MIRA term we have proven our ability to develop impactful technology and advance innovative science on several fronts. In this second term, we aim show sustainability of our research endeavors, solidifying our niches, maintaining high productivity, and ultimately producing knowledge and tools to benefit the scientific community.