Controlled, Sequential Folding of Structured RNAs

结构化 RNA 的受控顺序折叠

• 批准号: 1516896
• 负责人: Craig Martin
• 金额: $ 65.56万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1516896&HistoricalAwards=false
• 关键词: Controlled; Sequential; Folding; Structured; RNAs

Ribonucleic acid polymers (RNA) are similar to proteins in that they can fold into defined three-dimensional shapes that are important for their function. RNAs have many functions in the cell in addition to coding for protein synthesis, and some long non-coding RNAs are now known to fold into structures that possess protein-like functions. This project will develop novel methods that will allow researchers to understand the complex process of RNA folding in biology, and to control this process to produce designed RNA molecules with desired three-dimensional shapes for use in synthetic biology. The project has the potential to revolutionize the study of RNA folding, providing a better understanding of how RNAs fold in living cells. To bring an appreciation of such biomolecular structures to the general public, the project will expand the "Molecular Playground," a system that projects animated, architectural scale molecular images in public spaces, with which the public can directly interact through a simple wave of the hand (a passerby can, for example, "grab" and rotate the molecule to see it from all sides). Specifically, the project will facilitate installation of inexpensive exhibits in middle and high schools, including those serving groups underrepresented in science, with an aim towards exciting young people towards the exploration of STEM fields.Sequential, cotranscriptional folding of long noncoding RNAs (ncRNA), often accompanied by programmed RNA polymerase pausing, is now appreciated to be critical to proper ncRNA function. Current in vitro RNA folding studies do not mimic the resulting directed topological constraints. Using toehold mediated strand displacement this proposal will develop a novel system that allows controlled, sequential release of RNA from an unstructured RNA-DNA duplex, modeling sequential, cotranscriptional folding. Breaking the exogenous displacing DNA strand into two segments or introducing mismatches in it allows the introduction of controlled pausing in folding. Initial characterization of the system will follow folding of fluorescent aptamers, and later studies will be directed at understanding folding of the coenzyme B12 riboswitch, a system known to require polymerase pausing. Critically, perturbations will not be introduced into the RNA itself, but will be engineered only into the displacing strand, providing a native fold in all cases. This novel approach has the potential to revolutionize the study of RNA folding, providing a better understanding of how ncRNAs fold in vivo, and providing new tools for synthetic biology.

核糖核酸聚合物 (RNA) 与蛋白质相似，它们可以折叠成对其功能很重要的定义的三维形状。除了编码蛋白质合成外，RNA 在细胞中还具有许多功能，现在已知一些长非编码 RNA 可以折叠成具有蛋白质样功能的结构。该项目将开发新颖的方法，使研究人员能够了解生物学中 RNA 折叠的复杂过程，并控制该过程以生产具有所需三维形状的设计 RNA 分子，用于合成生物学。该项目有可能彻底改变 RNA 折叠的研究，让人们更好地了解 RNA 在活细胞中的折叠方式。为了让公众了解这种生物分子结构，该项目将扩展“分子游乐场”，这是一个在公共空间投影动画、建筑规模的分子图像的系统，公众可以通过简单的挥手直接与该图像互动（例如，路人可以“抓住”并旋转分子以从各个侧面看到它）。具体来说，该项目将促进在初中和高中安装廉价的展览，包括那些在科学领域中代表性不足的服务群体，目的是激发年轻人对 STEM 领域的探索。长非编码 RNA (ncRNA) 的顺序共转录折叠，通常伴随着程序化的 RNA 聚合酶暂停，现在被认为对于正常的 ncRNA 功能至关重要。目前的体外 RNA 折叠研究并未模拟由此产生的定向拓扑约束。该提案将利用立足点介导的链置换开发一种新型系统，允许从非结构化 RNA-DNA 双链体中受控、顺序释放 RNA，模拟顺序、共转录折叠。将外源置换 DNA 链断裂成两个片段或在其中引入错配，可以在折叠过程中引入受控暂停。该系统的初步表征将遵循荧光适体的折叠，后续研究将旨在了解辅酶 B12 核糖开关的折叠，这是一种已知需要聚合酶暂停的系统。重要的是，扰动不会被引入 RNA 本身，而只会被工程化到置换链中，从而在所有情况下提供天然折叠。这种新方法有可能彻底改变 RNA 折叠的研究，让人们更好地了解 ncRNA 在体内如何折叠，并为合成生物学提供新工具。