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，因为它们可以折叠成定义​​的三维形状，这些形状对其功能很重要。除编码蛋白质合成外，RNA在细胞中具有许多功能，现在已知一些长的非编码RNA折叠成具有蛋白质样功能的结构。该项目将开发新的方法，使研究人员能够理解生物学中RNA折叠的复杂过程，并控制此过程，以产生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，从而更好地了解活细胞中RNA的折叠方式。为了向公众欣赏这种生物分子结构，该项目将扩展“分子游乐场”，该系统在公共空间中投射动画，建筑尺度的分子图像，公众可以直接通过简单的手来互动（例如，一个路人可以，例如，一个路人可以“抓取”并旋转分子，从所有侧面旋转它）。具体而言，该项目将有助于在中学和高中安装廉价的展览，包括那些在科学方面代表性不足的人，其目的是令人兴奋的年轻人朝着探索STEM领域的探索。对长期不编码的RNA（NCRNA）的共同折叠（NCRNA），通常会伴随着编程的RNA Polymase unna，以备受良好的功能。当前的体外RNA折叠研究不能模仿所得的定向拓扑约束。使用toehold介导的链位移该建议将开发出一种新型系统，该系统允许从非结构化的RNA DNA双链体中控制RNA的顺序释放，对顺序，共表折叠进行建模。将DNA链分为两个段或引入不匹配的外源性位移，从而引入了受控的暂停折叠。该系统的初始表征将遵循荧光体适体的折叠，后来的研究将致力于了解辅酶B12核糖开关的折叠，该系统已知需要聚合酶暂停。至关重要的是，不会将扰动引入RNA本身，而只能设计到位移链中，在所有情况下都提供本地折叠。这种新颖的方法有可能彻底改变RNA折叠的研究，从而更好地了解NCRNA在体内如何折叠，并为合成生物学提供新的工具。