Controlled, Sequential Folding of Structured RNAs

核糖核酸聚合物（RNA）与蛋白质相似，它们可以折叠成明确的三维形状，这对它们的功能很重要。除了编码蛋白质合成外，rna在细胞中还有许多功能，现在已知一些长链非编码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，使研究人员能够理解生物学中RNA折叠的复杂过程，并控制这一过程，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，使人们更好地了解RNA在活细胞中的折叠方式。为了让公众欣赏这种生物分子结构，该项目将扩大“分子游乐场”，这是一个在公共空间中投射动画的建筑尺度分子图像的系统，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）。具体而言，该项目将促进在初中和高中（包括为科学领域代表性不足的群体服务的学校）安装廉价展品，目的是激发年轻人探索STEM领域。长链非编码RNA （ncRNA）的顺序、共转录折叠，通常伴随着程序性RNA聚合酶暂停，现在被认为是ncRNA正常功能的关键。目前的体外RNA折叠研究并没有模拟由此产生的定向拓扑限制。利用支点介导的链位移，本提案将开发一种新的系统，允许从非结构化RNA- dna双工中控制RNA的顺序释放，模拟顺序的共转录折叠。将外源性取代的DNA链断裂成两段或在其中引入错配，可以在折叠中引入受控暂停。该系统的初步表征将遵循荧光适体的折叠，随后的研究将针对理解辅酶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RNA本身，而只会被改造到置换链中，在所有情况下都提供天然折叠。这种新方法有可能彻底改变RNA折叠的研究，更好地了解ncRNAs在体内如何折叠，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.