CAREER: A Novel Framework for Measuring and Engineering Twisting and Writhing in DNA

基因是每个活细胞内生物密码的基本单位。每个细胞的基因组都有数千个基因，以不同的空间模式排列，产生复杂的，协调的遗传程序。每当一个基因被打开时，DNA的扭曲的双螺旋结构必须被解螺旋，以暴露单链DNA用于RNA合成（转录）。当成千上万的基因同时转录时，DNA的错位扭曲在基因组中穿行，形成了一个活生生的、流动的DNA扭曲景观。该项目将开发新的生物技术，以揭示DNA持续扭曲的动力学如何改变细胞活性和细胞命运。总体目标是了解遗传程序的不同空间排列与DNA扭曲的流体景观之间的功能关系。使用合成生物学，实验生物物理学和控制理论的技术相结合，这项工作将使研究人员能够在实验上可视化和建模本地化的DNA扭曲，与基因转录相一致。这些结果有望为如何在空间上设计基因的合成邻域以及最终可用于生物技术的合成基因组提供实际的理解。研究机会将提供给研究生和本科生。此外，该项目还将与加州圣伊内斯、圣玛利亚和隆波克学区的教师合作，开发课程、实践培训讲习班、教育视频和互动真人大小的空间模型，以促进对DNA可编程性和动力学的理解。生物学家以前曾研究过相对较短的DNA链扭曲的迁移动力学。该项目旨在同时可视化和整合DNA扭曲和基因转录过程的模型，这些模型在历史上一直在不同的时间尺度上分别进行研究。PI最近功能化和集成的磁镊子荧光显微镜系统与无细胞反应器在小体积毛细管。这个系统，结合一系列最近开发的遗传电路和报告结构，将用于研究DNA扭曲和扭曲对转录的影响。同时，在线性和环状DNA中形成高阶DNA结，如plectonemes，将被可视化。这些都是新的体外方法，用于测量局部超螺旋密度如何改变或控制附近的基因转录。该项目还将开发新的体内生物传感器，测量活细胞中环状DNA的局部扭曲和扭曲程度，将体内超螺旋与附近的转录活性联系起来。此外，生物物理模型将被开发，以描述如何空间分布，运输和绝缘的本地化超螺旋控制转录的DNA.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。

Genes are the basic units of biological code inside of every living cell. The genome of every cell has thousands of genes, arranged in distinct, spatial patterns that give rise to complex, coordinated genetic programs. Each time a gene is turned on, the twisted, double helical structure of DNA must be uncoiled, to expose single-stranded DNA for RNA synthesis (transcription). As thousands of genes are simultaneously transcribed, the displaced twists of the DNA travel across the genome, forming a living, fluid landscape of DNA writhing. This project will develop new biotechnology to reveal how the dynamics of persistent twisting in DNA alters cellular activity and cell fate. The overarching goal is to understand the functional relationship between different spatial arrangements of genetic programs and the fluid landscape of DNA twisting. Using a combination of techniques from synthetic biology, experimental biophysics, and control theory, the work will allow researchers to experimentally visualize and model localized DNA twisting, in concert with gene transcription. The results are expected to provide practical understanding of how to spatially design synthetic neighborhoods of genes and ultimately, synthetic genomes that could be used in biotechnology. Research opportunities will be provided for graduate and undergraduate students. In addition, in collaboration teachers in the Santa Ynez, Santa Maria and Lompoc school districts in California, the project will develop curricula, hands-on training workshops, educational videos, and interactive life-sized spatial models to promote understanding of the programmability and the dynamics of DNA. Biologists have previously studied the migratory dynamics of twisting in relatively short strands of DNA. This project aims to simultaneously visualize and integrate models for the processes of DNA twisting and gene transcription, which historically have been studied separately on different timescales. The PI has recently functionalized and integrated a magnetic tweezer fluorescence microscopy system with cell-free reactors in small-volume capillary tubes. This system, combined with an array of recently developed genetic circuits and reporter constructs, will be used to study of effects of DNA writhing and twisting on transcription. Simultaneously, the formation of higher-order DNA knots in linear and circular DNA, such as plectonemes, will be visualized. These are new in vitro methods for measuring how local supercoiling density alters or controls nearby gene transcription. The project also will develop new in vivo biosensors that measure the degree of localized twist and writhe in circular DNA in living cells, to link in vivo supercoiling to nearby transcriptional activity. In addition, biophysical models will be developed to describe how the spatial distribution, transport, and insulation of localized supercoiling controls transcription in DNA.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.