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

项目总结 研发项目1.样本阶段：分离和保存大分子层次结构的工具 任何给定的大分子都可以与其他大分子发生稳定的、动态的或瞬时的相互作用。这些 相互作用形成一个层次化的网络，包括细胞互动体；而这个整个网络是 被其他络合物组成的大分子环境包围，这些络合物相互碰撞，可以形成邻近的相互作用。 Tr&d1代表了我们流水线的第一个模块，其中的挑战是高保真地保存和 分离任何感兴趣的大分子的本地天然细胞内大分子环境。要访问 然后将一个大分子及其相互作用分子在它们天然的层级组装中分离出来，我们必须打破 牢房。然而，这扰乱了细胞的大规模组织；以及稀释、环境变化、 正常分离成分的混合，能量再生的损失，以及暴露在降解物中 酶导致分解、扰乱和聚集。所有这些因素都会随着时间的延长而恶化 细胞破坏和组件的分析性分离之间的关系。我们的解决方案是既要快速捕获，又要 尽可能高效地使用这些程序集，并尽可能地保留它们的本机状态。要完成 为此，我们将生产用于超高效检测和捕获组件的优化试剂。我们会 继续为广泛使用的现有标签和未添加标签的目标开发诱饵 很实用。与其他亲和捕获试剂一样，我们的中心在其 生成纳米体的平台，亲和力捕获方法的突出诱饵。我们还将继续 开发方法忠实地保存自然的细胞相互作用，以便随后分离内源 复合体。我们是通过将细胞内的复合体冻结在“暂停”状态来实现这一点的 动画“，然后冷冻以访问细胞的复合体，而它们仍然是冰冻的；这些复合体必须 快速、清洁和方便地从周围的细胞裂解物中分离出来，以保存 该复合体具有理想的特性，同时最大限度地减少了对其他污染物的粘附性。 在这里，我们将开发快速优化这些亲和力捕获的方法，它们都可以移植到(I)工作台 可为任何其他研究人员进行扩展，以及(Ii)易于访问、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，我们正专注于 建立简便、可靠、广泛使用的高效快速分离的试剂和方法 大分子复合体。我们希望扩大这一进展并开发使能的新技术，以使 任何研究人员都可以方便而全面地获取两者动态交互中的信息 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.