A novel in vitro microscopy suite to elucidate intracellular transport and conformational dynamics of nucleic acids

项目摘要 该项目的目标是开发一个强大的平台，结合尖端的显微镜方法，图像分析， 算法，微流体和大分子合成技术，以全面表征 核酸在良好控制的、随时间变化的仿生环境中。长远的目标是将核酸 细胞内环境的动态，重点是大的裸DNA，特别是由最近工程化的核酸 用于基因治疗的酸性复合物和最近发现的大型病毒的基因组。理解的主要障碍 细胞内核酸转运是大分子和网络的压倒性的复杂性和多样性， 拥挤细胞虽然许多研究已经探索了拥挤环境中的大分子动力学，但实验 主要是在：单分散，稳态体外拥挤系统，无法复制复合物 细胞内环境;或高度异质性的，动态的体内系统，这提供了不完整的理解， 大分子成分和性能负责观察运输。建议综合实验 工具集将被设计为弥合这一差距，并直接耦合单大分子运输到（i）构象 动力学，（ii）系综运输，和（iii）拥挤的环境属性。应用这一方法的关键第一步 细胞的平台是设计和利用良好控制的体外细胞骨架网络，可以精确地调整超过一个 宽的参数空间，以产生体内观察到的丰富的DNA动力学，并将观察到的动力学与可调的 变量包括DNA和细胞骨架的属性。环境和DNA参数将是不同的， 由此产生的DNA和网络动力学将使用一个平台，包括（a）光片显微镜， 探测单分子和整体DNA动力学以及环境特性的方法，（B）仿生 细胞骨架环境包括不同量的（i）肌动蛋白和（ii）微管，（c）自制的微流体 灌注室，用于实时原位调节（i）和（ii）的聚合状态，和（d）定制工程 不同长度和拓扑结构的荧光标记DNA分子。目前的研究将集中在优化和 传播这个强大的平台，SLAMMTAP（时空光片辅助多尺度大分子 运输分析探针），并证明其效用和适用性，以健康科学研究人员通过确定关键 DNA（大小、拓扑结构）和细胞骨架环境（浓度、刚度、交联密度， 时空异质性），导致单分子运输，构象动力学和集体扩散 模拟体内观察到的复杂现象的DNA。最终，在体外和体内研究的DNA动力学允许 该项目开发的技术将揭示病毒基因组如何穿越拥挤的细胞质， 帮助指导研究人员设计具有最佳疗效的含DNA或RNA的基因疗法。

Project Summary The goal of this project is to develop a powerful platform that combines cutting-edge microscopy methods, image analysis algorithms, microfluidics, and macromolecular synthesis techniques to comprehensively characterize the dynamics of nucleic acids in well-controlled, time-varying biomimetic environments. The long-term vision is to characterize nucleic acid dynamics in the intracellular milieu, with a focus on large naked DNA, specifically motivated by recently engineered nucleic acid complexes for gene therapy and genomes of recently discovered macroviruses. The primary hindrance to understanding intracellular nucleic acid transport is the overwhelming complexity and diversity of macromolecules and networks that crowd cells. While many studies have explored macromolecular dynamics in crowded environments, experiments have largely been carried out either in: monodisperse, steady-state in vitro crowded systems, that fail to replicate the complex intracellular environment; or highly heterogeneous, dynamic in vivo systems, which offer incomplete understanding of the macromolecular components and properties responsible for observed transport. The proposed comprehensive experimental toolset will be designed to bridge this gap, and to directly couple single-macromolecule transport to (i) conformational dynamics, (ii) ensemble transport, and (iii) crowded environment properties. The vital first step towards applying this platform to cells is to design and utilize well-controlled in vitro cytoskeleton networks that can be precisely tuned over a wide parameter space to generate the rich DNA dynamics observed in vivo and to couple observed dynamics to tunable variables that include properties of both DNA and cytoskeleton. Environmental and DNA parameters will be varied and the resulting DNA and network dynamics will be measured using a platform consisting of (a) light-sheet microscopy with methods to probe single-molecule and ensemble DNA dynamics as well as environmental properties, (b) biomimetic cytoskeleton environments comprised of varying amounts of (i) actin and (ii) microtubules, (c) home-built microfluidic perfusion chambers for in situ modulation of polymerization states of (i) and (ii) in real-time, and (d) custom-engineered fluorescent-labeled DNA molecules of varying lengths and topologies. The current research will focus on optimizing and disseminating this robust platform, SLAMMTAP (Spatiotemporal Light-sheet Assisted Multiscale Macromolecular Transport Analysis Probe), and proving its utility and applicability to health-science researchers by identifying the key characteristics of both DNA (size, topology) and cytoskeleton environments (concentration, stiffness, crosslinking density, spatiotemporal heterogeneities) that lead to single-molecule transport, conformational dynamics and collective diffusion of DNA that mimic complex phenomena observed in vivo. Ultimately, in vitro and in vivo studies of DNA dynamics allowable by the techniques developed in this project will shed light on how viral genomes traverse the crowded cytoplasm and will help guide researchers to engineer DNA- or RNA-containing gene therapies with optimal efficacy.