Ultrasensitive acoustic reporter proteins with engineered rupture-resistant shells

具有工程抗破裂外壳的超灵敏声学报告蛋白

基本信息

批准号：
10511817
负责人：
George J Lu
金额：
$ 22.9万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-17 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10511817
关键词：
Acoustics Adoption Alanine Amino Acids Animal Model Award Benchmarking Biological Process Biology Biomedical Research Caenorhabditis elegans Carbamates Cell Therapy Cells Chemicals Chemistry Detection Diagnosis Diagnostic Disease Elements Engineering Ensure Escherichia coli Evaluation Exploratory/Developmental Grant Funding Mechanisms Gases Gene Expression Generations Genes Genetic Code Goals Human Image Light Lysine Mammalian Cell Maps Mass Spectrum Analysis Measurement Measures Mechanics Methods Microbe Microbubbles Modeling Monitor Movement Mutagenesis Names Nanostructures Organism Outcome Penetration Phase Process Property Protein Engineering Proteins Protocols documentation Reporter Reporter Genes Research Personnel Resistance Rodent Rodent Model Rupture Scientist Signal Pathway Site Solid Stress Structural Models Structure Technology Tensile Strength Therapeutic Time Tissues Ultrasonography Vesicle Visualization base cellular imaging crosslink crystallinity design disease diagnosis fluorescence imaging hemodynamics high risk imaging agent imaging biomarker improved in vitro testing innovation interdisciplinary approach mechanical properties new technology non-invasive imaging particle pressure screening simulation submicron synthetic biology targeted imaging technology development time use tool ultrasound

项目摘要

Project Summary / Abstract The ability to image specific gene expression and cellular signaling pathways has long been a powerful tool for scientists, and the methods to achieve this goal, such as the fluorescent reporter genes, are widely used nowadays across different fields of biomedicine. However, the penetration depth of light has limited the use of fluorescent reporter genes in animal models and cell-based therapies in humans. To this end. a group of gas- filled protein nanostructures named gas vesicles (GVs) were recently introduced as the acoustic reporter genes and enabled, for the first time, the use of ultrasound imaging to visualize specific gene expression in cells located at centimeter-deep tissue. GVs are sub-micron particles originally evolved in photosynthetic microbes to achieve cellular flotation, and expectedly, these wildtype GVs do not possess the correct properties that can maximize their detection by ultrasound. This limitation on imaging sensitivity is perhaps the biggest hurdle currently facing the application of the first-generation acoustic reporter genes. In this proposal, we aim to introduce the second- generation acoustic reporter genes by innovating the mechanical properties of the protein shell. In Aim 1, we will leverage the Genetic Code Expansion technology to achieve site-specific crosslinking among the monomeric shell proteins. This is to recognize that the inward buckling of the protein shell holds the key to the sensitive detection of these acoustic protein nanostructures but often leads to the rupture of the wildtype GV shell, and a systematic crosslinking will substantially increase the tensile strength of these protein shells. Specifically, we will systematically search sites that allow the incorporation of non-canonical amino acid and lysine for proximity- induced chemistry, and this search will be performed under the rational guidance of the structural models. In parallel to Aim 1, we will develop finite-element modeling and acoustic measurement method in Aim 2 to understand the effect of crosslinking on the mechanical properties of the shell, which will establish benchmark values and aims for the design of the protein nanostructures. In Aim 3, we seek to establish sensitivity enhancement of these rupture-resistant acoustic protein nanostructures in a rodent model. Our interdisciplinary approach merges synthetic biology, chemical biology, acoustics, and solid mechanics, and if successful, this high-risk high-gain project will lead to a new generation of ultrasensitive acoustic reporter genes that broaden the technology to many therapeutic and diagnostic applications, especially in the functional tracking of cell-based therapies and targeted imaging of biomarkers.

项目概要/摘要对特定基因表达和细胞信号通路进行成像的能力长期以来一直是一个强大的工具科学家们，实现这一目标的方法，例如荧光报告基因，被广泛使用目前已涉足生物医学的不同领域。然而，光的穿透深度限制了其使用动物模型中的荧光报告基因和人类细胞疗法。为此。一组气体- 最近引入了名为气体囊泡（GV）的填充蛋白质纳米结构作为声学报告基因并首次使用超声成像来可视化位于细胞中的特定基因表达在厘米深的组织中。 GV 是亚微米颗粒，最初是在光合微生物中进化来实现细胞浮选，并且预期这些野生型 GV 不具备可以最大化的正确特性通过超声波检测它们。成像灵敏度的限制可能是目前面临的最大障碍第一代声学报告基因的应用。在这个提案中，我们的目标是引入第二个—— 通过创新蛋白质壳的机械特性来生成声学报告基因。在目标 1 中，我们将利用遗传密码扩展技术实现单体之间的位点特异性交联壳蛋白。这是为了认识到蛋白质外壳的向内屈曲是敏感的关键检测这些声学蛋白纳米结构，但通常会导致野生型 GV 外壳的破裂，并且系统交联将大大增加这些蛋白质壳的拉伸强度。具体来说，我们将系统地搜索允许掺入非规范氨基酸和赖氨酸的位点以进行接近- 诱导化学，这种搜索将在结构模型的合理指导下进行。在与目标 1 并行，我们将在目标 2 中开发有限元建模和声学测量方法，以了解交联对壳机械性能的影响，这将建立基准蛋白质纳米结构设计的价值和目标。在目标 3 中，我们寻求建立敏感性在啮齿动物模型中增强这些抗破裂声学蛋白质纳米结构。我们的跨学科方法融合了合成生物学、化学生物学、声学和固体力学，如果成功的话，这个高风险高收益项目将产生新一代超灵敏声学报告基因，拓宽该技术适用于许多治疗和诊断应用，特别是基于细胞的功能跟踪生物标志物的治疗和靶向成像。