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

RNA是一类在基因表达中起核心作用的生物合成分子，基因表达是利用DNA中储存的遗传信息制造功能蛋白质的过程。RNA在基因表达中的功能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结构变化（它们的动态重排）的能力。凭借这一奖项，化学部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结构的动态变化如何控制人类基因的表达。Gonzalez博士和Nuckolls博士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力学。值得注意的是，smFET方法允许研究RNA动力学，而不需要化学修饰RNA或施加侵入性外力。对基于RNA的结构开关如何控制基因表达的更深入理解影响了作用于重要生物分子靶标和/或功能的治疗剂的发现和开发。此外，该项目通过利用纽约市现有的外展计划，增加了妇女和代表性不足的少数民族在STEM中的参与。开发工具包，以展示K-12社区外展的核酸结构和单分子生物物理学，同时开发教育模块，在当地高中教授化学，生物学和计算机科学的综合概念。Gonzalez博士和Nuckolls博士的研究团队使用smFET研究了RNA茎环（RNA结构的基本类别）折叠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的动力学机制。所得到的模型用于确定机制，通过该机制，在人前体信使RNA（前mRNA）内发现的RNA茎环的茎中腺苷的N6位置的天然存在的转录后甲基化调节该RNA茎环的动力学，作为控制前mRNA剪接的手段。具体而言，所提出的研究表征了该茎环的折叠和动力学，并测试了腺苷的转录后N6甲基化改变茎环动力学的假设（即，打开“开关”），从而揭开指导前体mRNA差异剪接的RNA结合蛋白的结合位点。这些研究的结果提供了一个通用模型的功能RNA茎环为基础的构象开关和详细了解的机制，通过转录后修饰的茎环控制的选择性剪接的pre-mRNA.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

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.