Label-Free, High-Time Resolution, Single-Molecule Studies of RNA Folding and Dynamics

RNA是一类生物合成分子，在基因表达中起着核心作用，基因表达是利用储存在DNA中的遗传信息制造功能性蛋白质的过程。rna如何在基因表达中发挥作用取决于它们特定而复杂的三维形状或结构，以及它们经历精确定时和执行的结构变化（它们的动态重排）的能力。有了这个奖项，化学部门的生命过程化学项目将资助哥伦比亚大学的Ruben Gonzalez博士和Colin Nuckolls博士研究RNA如何折叠和/或进行结构重排，以及特定RNA结构“开关”的动力学如何控制人类基因的表达。Drs。Gonzalez和Nuckolls的研究小组使用基于碳纳米管的场效应晶体管（smfet）来跟踪单个RNA分子在三维空间和时间上的详细动态。值得注意的是，smFET方法允许研究RNA动力学，而无需对RNA进行化学修饰或施加侵入性外力。更深入地了解基于rna的结构开关如何控制基因表达，将影响作用于重要生物分子靶点和/或功能的治疗方法的发现和发展。此外，该项目通过利用纽约市现有的外展项目，提高了女性和代表性不足的少数族裔对STEM的参与。开发了用于在K-12社区外展中展示核酸结构和单分子生物物理学的工具包，同时开发了用于在当地高中教授化学、生物和计算机科学综合概念的教育模块。利用smfet， dr。Gonzalez和Nuckolls的研究小组研究了RNA茎环（RNA结构的一个基本类别）折叠的动力学机制，并以无与伦比的分子、空间和时间分辨率进行功能重要的动态结构重排。由此产生的模型用于确定在人类前体信使RNA （pre-mRNA）中发现的RNA茎环茎中腺苷N6位置的自然发生转录后甲基化调节该RNA茎环动力学的机制，作为控制pre-mRNA剪接的手段。具体来说，提出的研究表征了这种茎环的折叠和动力学，并验证了这样一种假设，即转录后，腺苷的N6甲基化改变了茎环的动力学（即打开“开关”），从而揭示了指导前mrna差异剪接的rna结合蛋白的结合位点。这些研究结果为基于RNA茎环的构象开关的功能提供了一个通用模型，并详细了解了茎环的转录后修饰控制前mrna的选择性剪接的机制。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。

RNA is a class of biosynthetic molecules that plays central roles in gene expression, the process of using the genetic information stored in DNA to make functional proteins. How RNAs function in gene expression is dependent on their specific and complex three-dimensional shapes or structures and on their ability to undergo precisely timed and executed structural changes (their dynamic rearrangements). With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Ruben Gonzalez and Dr. Colin Nuckolls from Columbia University to study how RNAs fold and/or undergo structural rearrangements and how the dynamics of a particular RNA structural ‘switch’ controls the expression of a human gene. Drs. Gonzalez’s and Nuckolls’ research team uses carbon nanotube-based field-effect transistors (smFETs) to follow the detailed dynamics of individual RNA molecules in three-dimensional space and across time. Notably, the smFET method allows the study of RNA dynamics without the need to chemically modify the RNA or apply invasive external forces. A deeper understanding of how RNA-based structural switches function to control gene expression impacts the discovery and development of therapeutics that act on important biomolecular targets and/or functions. In addition, the project increases participation by women and underrepresented minorities in STEM by leveraging existing outreach programs in New York City. Kits are developed to demonstrate nucleic acid structures and single-molecule biophysics in K-12 community outreach, while education modules are developed to teach integrated concepts of chemistry, biology, and computer science at local high schools. Using smFETs, Drs. Gonzalez’s and Nuckolls’ research team investigate the kinetic mechanisms with which RNA stem-loops, a fundamental class of RNA structures, fold and undergo functionally important, dynamic structural rearrangements at unparalleled molecular, spatial, and temporal resolutions. The resulting model is used to determine the mechanism through which the naturally occurring, post-transcriptional methylation of the N6 position of an adenosine in the stem of a RNA stem-loop found within a human precursor messenger RNA (pre-mRNA) regulates the dynamics of this RNA stem-loop as a means for controlling the splicing of the pre-mRNA. Specifically, the proposed studies characterize the folding and dynamics of this stem-loop and tests the hypothesis that the post-transcriptional, N6 methylation of the adenosine alters the dynamics of the stem-loop (i.e., throws the ‘switch’ on) so as to unmask the binding site of an RNA-binding protein that directs the differential splicing of the pre-mRNA. The results of these studies provide a general model for the function of RNA stem-loop based conformational switches and a detailed understanding of the mechanism through which a post-transcriptional modification of a stem-loop controls the alternative splicing of a pre-mRNA.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.