TR&D Project 1. The Sample Stage: Tools for Isolating and Preserving Macromolecular Hierarchies

项目摘要 TR&D项目1.样品台：分离和保存大分子等级的工具 任何一种大分子都可以与其它大分子发生稳定的、动态的或瞬时的相互作用。这些 相互作用形成了一个分层网络，包括细胞相互作用组;整个网络是 被其他复合物的大分子环境所包围，这些复合物与它相竞争，并可以形成邻位相互作用。 TR&D1代表了我们管道的第一个模块，其中的挑战是保持高保真度， 分离任何目标大分子的局部天然细胞内大分子环境。访问 然后将一个大分子及其相互作用物分离在它们的天然层次结构中，我们必须打破 牢房然而，这破坏了细胞的大规模组织;稀释，环境变化， 通常分离的组分的混合、能量再生的损失和暴露于降解性物质 酶引起分解、混乱和聚集。所有这些因素随着时间的延长而恶化 细胞破裂和分析分离的组合体之间的关系。我们的解决方案是既能快速捕获， 这些程序集尽可能高效地运行，并尽可能地保持它们的原生状态。完成 因此，我们将生产用于超高效检测和捕获组装体的优化试剂。我们将 继续为广泛使用的现有标签和不添加标签的目标开发诱饵 实用.以及其他亲和捕获试剂，我们的中心提供了一个独特的BTRR服务， 平台产生nanobodies，优秀的诱饵亲和捕获方法。我们还将继续 开发忠实地保留天然细胞相互作用的方法，用于随后分离内源性 配合物我们通过将复合物冻结在细胞内的“悬浮”状态来实现这一点。 动画”，然后冷冻研磨，以访问细胞的复合物，而他们仍然冻结;这些复合物必须是 快速、干净和方便地从其周围的细胞裂解物中分离出来， 复合物的所需特性，同时使附加污染材料的粘附最小化。 在这里，我们将开发用于快速优化这些亲和捕获的方法，这些方法都是便携式的（i）工作台 规模为任何其他研究人员和（ii）随时访问，负担得起的机器人平台，以最大限度地提高吞吐量 和再现性。由于这些技术必须随时可供任何实验室使用， 建立简单、稳健和广泛使用的试剂和方法， 大分子复合物我们希望扩大这一进展，并开发新的技术， 任何研究人员都可以轻松全面地访问两者动态相互作用组中的信息 正常和病态的生命系统。

PROJECT SUMMARY TR&D Project 1. The Sample Stage: Tools for Isolating and Preserving Macromolecular Hierarchies Any given macromolecule may make stable, dynamic or transient interactions with other macromolecules. These interactions form a hierarchical network, comprising the cellular interactome; and this whole network is surrounded by a macromolecular milieu of other complexes that jostle with it, and can form vicinal interactions. TR&D1 represents the first module of our pipeline, where the challenge is to preserve with high fidelity and isolate the local native intracellular macromolecular environment of any macromolecule of interest. To access and then isolate a macromolecule and its interactors in their native hierarchical assemblies, we must break open the cell. However, this disrupts the cell’s large-scale organization; and factors such as dilution, change in milieu, the intermingling of normally segregated components, loss of energy regeneration, and exposure to degradative enzymes cause disassembly, scrambling and aggregation. All these factors worsen as the time lengthens between cell disruption and analytic isolation of the assemblies. Our solution is both to capture as rapidly and efficiently as possible these assemblies, and preserve their native state as much as possible. To accomplish this, we will produce optimized reagents for the ultra-efficient detection and capture of assemblies. We will continue to develop baits for both widely-used existing tags and targets for which the addition of tags are not practical. As well as other affinity capture reagents, our Center provides offers a unique BTRR service in its platform to generate nanobodies, outstanding baits for affinity capture approaches. We will also continue to develop methods for faithfully preserving native cellular interactions for subsequent isolation of endogenous complexes. We accomplish this by freezing complexes in place inside the cell in a state of “suspended animation”, then cryomilling to access the cell’s complexes while they are still frozen; these complexes must be isolated quickly, cleanly and conveniently from its surrounding cellular lysate in such a way as to preserve the desired characteristics of the complex while minimizing the adherence of additional contaminating materials. Here, we will develop means for rapidly optimizing these affinity captures, which are both portable to (i) bench scale for any other researcher and (ii) readily accessible, affordable robotic platforms to maximize throughput and reproducibility. Because these technologies must be readily accessible to any laboratory, we are focusing on establishing simple, robust and widely used reagents and approaches for the efficient and rapid isolation of macromolecular complexes. We wish to extend this progress and develop enabling new technologies to allow any researcher to readily and comprehensively access the information in the dynamic interactomes of both normal and diseased living systems.