MFB: Continuous evolution of RNAs with novel functions in mammalian cells

功能性RNA分子在生物研究、生物工程和医学方面具有巨大的应用潜力。重要的是，现在可以很容易地识别rna，它们可以结合几乎任何其他感兴趣的生物分子，并显示出独特的功能，包括催化化学反应、荧光等。但是有一个主要的挑战阻碍了进展——RNA结构对局部环境非常敏感。目前鉴定功能性rna的策略几乎都是在试管中进行的，大多数产生的功能性rna也只在试管中发挥作用，不幸的是，当翻译到生物环境中时就失败了。该项目旨在提供并优化一个新的平台，该平台将能够在哺乳动物细胞环境中直接快速、可靠地识别功能性rna。该方法涉及应用下一代策略来实现细胞中实验室时间尺度的RNA进化，从而实现所需RNA功能的有效选择。由于这些新的rna是在细胞环境中被鉴定出来的，因此它们将在生物环境中始终发挥作用——不像使用目前基于试管的方法发现的功能性rna。在取得这一进展的同时，该项目将为博士生培训提供机会，并使一个正在进行的、成功的高中实习项目得到实质性扩展。这项工作的首要假设是，基于细胞内的定向进化可以解决阻碍功能性RNA领域的环境敏感折叠和结构挑战。该研究旨在提供一个定制的、定制设计的哺乳动物噬菌体辅助连续进化系统，以实现对哺乳动物细胞功能RNA适配体、变构激活剂和粘合RNA的稳健和有效鉴定。所开发的选择对将是高度模块化的，并且对于该领域的任何人都可以进化出感兴趣的功能性rna。由此产生的RNA- mpace平台旨在提供易用性、支持并行化、规模化和定向进化运动速度的策略，以及专门针对编码感兴趣RNA的基因进行突变的方法，以进一步加速功能性RNA的开发。这项研究的成功实施将使功能性rna在生物学中的巨大潜力最终得到释放。在研究应用方面，调节任何感兴趣的生物分子的活性将变得更加可行。对于医学应用，rna可能成为所有类型靶标的首选形式。关键的基本见解也将变得容易获得，特别是与细胞环境如何塑造RNA结构功能景观有关。这项工作由生物科学理事会的分子和细胞生物科学部（MCB）、数学和物理科学理事会的化学部以及MCB的系统和合成生物学集群共同支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Functional RNA molecules hold tremendous potential for applications in biological research, bioengineering, and medicine. Importantly, RNAs can now be readily identified that bind virtually any other biomolecule of interest and display unique functions, including enabling the catalysis of chemical reactions, fluorescence, and more. But there is a major challenge obstructing progress – RNA structure is incredibly sensitive to the local environment. Current strategies to identify functional RNAs almost universally proceed in test tubes, mostly yielding functional RNAs that also work well only in a test tube and unfortunately fail when translated into biological environments. This project seeks to deliver and optimize a new platform that will enable rapid and robust identification of functional RNAs directly in the mammalian cellular environment. The approach involves application of next-generation strategies to attain laboratory-timescale RNA evolution in cells, enabling efficient selection for desired RNA functions. Because the novel RNAs are identified within cellular milieus to begin with, they will consistently function in biological settings – unlike functional RNAs discovered using current test tube-based methods. Alongside this progress, the project will provide opportunities for PhD student training as well as enable substantive expansion of an ongoing, successful high school internship program.The overarching hypothesis of this work is that in-cell based directed evolution can solve the environment-sensitive folding and structure challenge that has hamstrung the functional RNA field. The research aims to deliver a bespoke, custom-designed mammalian phage-assisted continuous evolution system to enable the robust and efficient identification of mammalian cell-functional RNA aptamers, allosteric activators, and glue RNAs. The selection couples developed will be highly modular and generally useful for anyone in the field to evolve functional RNAs of interests. The resulting RNA-mPACE platform is intended to deliver ease-of-use, strategies to support parallelization, scaling, and speed of directed evolution campaigns, and methods to specifically target mutagenesis to the gene encoding the RNA of interest to further accelerate functional RNA development. Successful execution of this research will enable the enormous potential of functional RNAs in biology to finally be unleashed. For research applications, it will become far more feasible to modulate the activity of any biomolecule of interest. For medical applications, RNAs could become a preferred modality for all types of targets. Key fundamental insights will also become accessible, especially related to how cellular environments shape RNA structure-function landscapes.This work is jointly supported by the Division of Molecular and Cellular Biosciences (MCB) in the Directorate for Biological Sciences, by the Division of Chemistry in the Directorate for Mathematical and Physical Sciences, and by the Systems and Synthetic Biology cluster in MCB.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.