Acquisition of a Confocal Fluorescence Microscope to Image Synthetic Biomolecular Condensates

摘要 在此MIRA应用程序中的拟议工作利用了我长期以来的兴趣和专业知识， 基因编码的刺激响应肽聚合物。我的团队率先开发了 具有较低临界溶解温度（LCST）相的重组弹性蛋白样多肽（ELP 行为同时，我们还率先开发了高通量组装方法， 我们用它来创建现存最大的重组肽聚合物文库。 对它们的水相行为的表征导致发现了可以用于 对于从头使用的肽聚合物的设计，所述肽聚合物表现出LCST相行为和一类节枝弹性蛋白样结构。 多肽（RLP）表现出匡威的上临界溶液相变（UCST）相 行为在这项工作的基础上，我们将探讨本提案中的两个新领域。首先，我们将研究如何 我们可以通过以下设计概括生物材料所表现出的分级结构和性质： 部分有序聚合物（POP）-由无序多肽组成，嵌有周期性的 重复的二级结构基序-表现出温度触发的分级自组装成 宏观材料，模拟体内组织的结构蛋白质，如弹性蛋白网络。我们将 对新型POP的设计进行了系统的探索，以验证有序和 链段水平的无序是一种新的稳健的设计原理， 在许多长度尺度上的分级自组装。第二，我们将开发一个新的调查线， 通过翻译后修饰（PTM）的基因编码的生物杂交聚合物联合收割机 肽和非肽组分以产生表现出触发的、分级自组装的生物材料 变成宏观材料。在这一目标下，我们将扩大我们的初步工作，在体内豆蔻酰化的ELP 和UCST展示RLP，以研究我们是否可以将结构指导肽转化为豆蔻酰化 底物，以产生豆蔻酰化多肽，其中豆蔻酰化片段可以指导分级的自我- 组装整个结构。我们还将研究用胆固醇修饰ELP和RLP， 有可能直接自我组装，磷酸化，这将提供一个独特的触发自我， 组装件.在这两个领域仍有许多工作要做，因为我们对这些新领域的初步尝试只是暗示， 这些方法使新生物材料的分子设计具有巨大的可能性。工作 我们在此提出，有望产生具有有趣结构和性质的新生物材料， 在生物技术和医学中的应用。

Abstract The proposed work in this MIRA application leverages my long-standing interest and expertise in the design of genetically encoded stimulus-responsive peptide polymers. My group pioneered the development of recombinant elastin-like polypeptides (ELPs) that exhibit lower critical solution temperature (LCST) phase behavior. We have also, in parallel, pioneered the development of high-throughput methods for the assembly of highly repetitive genes that we used to create the largest extant library of recombinant peptide polymers. Characterization of their aqueous phase behavior led to the discovery of sequence heuristics that can be used for the de novo used design of peptide polymers that exhibit LCST phase behavior and a class of resilin-like polypeptides (RLPs) that exhibit the converse — upper critical solution phase transition (UCST) phase behavior. Building upon this work, we will explore two new areas in this proposal. First, we will investigate how we can recapitulate the hierarchical structure and properties exhibited by biological materials by the design of partially ordered polymers (POPs) —that consist of disordered polypeptides embedded with a periodically recurring secondary structure motif— that exhibit temperature triggered hierarchical self-assembly into macroscopic materials that mimic the in vivo organization of structural proteins like elastin networks. We will carry out a systematic exploration of the design of new POPs, to verify that the combination of order and disorder at the chain segment level is a new and robust design principle that will yield materials with hierarchical selfassembly across many length scales. Second, we will develop a new line of investigation on genetically encoded biohybrid polymers via post-translational modifications (PTMs) that precisely combine peptide and non-peptide components to create biomaterials that exhibit triggered, hierarchical self-assembly into macroscopic materials. In this aim, we will expand upon our initial work on in vivo myristoylation of ELPs and UCST exhibiting RLPs to investigate if we can convert structure-directing peptides into myristoylation substrates, to create myristoylated polypeptides where the myristoylated segment can direct hierarchical self- assembly of the entire construct. We will also investigate modification of ELPs and RLPs with cholesterol that has the potential to direct self-assembly, and phosphorylation, which will provide a unique trigger of self- assembly. Much remains to be done in both areas, as our preliminary foray into these new areas only hint at the enormous possibilities in the molecular design of new biomaterials enabled by these approaches. The work we propose herein promises to yield new biomaterials with interesting structures and properties with a host of applications in biotechnology and medicine.