Controlled, Sequential Folding of Structured RNAs

核糖核酸聚合物(RNA)类似于蛋白质，因为它们可以折叠成对其功能重要的定义的三维形状。除了编码蛋白质合成外，RNA在细胞中还有许多功能，现在已知一些长的非编码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，并控制这一过程，以生产设计的具有所需三维形状的RNA分子，用于合成生物学。该项目有可能给RNA折叠的研究带来革命性的变化，让人们更好地了解RNA在活细胞中是如何折叠的。为了给公众带来对这种生物分子结构的欣赏，该项目将扩大“分子游乐场”，这是一个在公共空间投射建筑规模的动画分子图像的系统，公众可以通过简单的挥手直接与之互动(例如，路人可以“抓住”并旋转分子从各个方面看到它)。具体地说，该项目将促进在初中和高中安装廉价的展品，包括那些在科学中代表不足的群体，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(NcRNA)，通常伴随着程序化的RNA聚合酶暂停，现在被认为是正常的ncRNA功能的关键。目前的体外RNA折叠研究并不能模拟由此产生的定向拓扑约束。利用Toehold介导链置换，这一提议将开发一种新的系统，允许从非结构化RNA-DNA双链中控制、顺序地释放RNA，模拟顺序的、共转录的折叠。将外源置换的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的暂停。该系统的初步表征将在荧光适配子折叠之后进行，稍后的研究将针对了解辅酶B12核糖开关的折叠，这是一个已知需要聚合酶暂停的系统。关键的是，扰动不会引入到RNA本身，而是只会被工程到置换链中，在所有情况下都提供自然折叠。这种新的方法有可能使RNA折叠的研究发生革命性的变化，更好地了解ncRNAs在体内是如何折叠的，并为合成生物学提供新的工具。

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.