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

• 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
• 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

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.

项目摘要 /摘要 长期以来，图像特定基因表达和细胞信号传导途径的能力一直是 科学家以及实现此目标的方法，例如荧光报告基因，广泛使用 如今，遍布不同生物医学领域。但是，光的穿透深度限制了 动物模型中的荧光报告基因和人类的基于细胞的疗法。为此。一组气体 最近引入了称为气囊泡（GVS）的填充蛋白纳米结构作为声学报告基因 并首次启用了超声成像以可视化的细胞中特定基因表达 在厘米深的组织时。 GV是最初在光合微生物中进化的亚微米颗粒以实现 细胞浮选，预计，这些野生型GV不具有最大化的正确特性 它们通过超声检测。对成像灵敏度的这种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。在此提案中，我们旨在介绍第二个 通过创新蛋白质壳的机械性能来产生声学报告基因。在AIM 1中，我们将 利用遗传代码扩展技术来实现单体中特定地点的交联 壳蛋白。这是为了认识到蛋白质壳的内向屈曲是敏感的钥匙 检测这些声学蛋白纳米结构，但通常会导致野生型GV壳破裂，A 系统的交联将大大提高这些蛋白质壳的拉伸强度。具体来说，我们会的 系统地搜索位点，该地点允许将非经典氨基酸和赖氨酸掺入以近似 诱导的化学反应，该搜索将在结构模型的合理指导下进行。在 与AIM 1平行，我们将在AIM 2中开发有限元建模和声学测量方法 了解交联对壳的机械性能的影响，这将建立基准 蛋白质纳米结构设计的价值和目标。在AIM 3中，我们试图建立灵敏度 在啮齿动物模型中，增强了这些抗破裂的声蛋白纳米结构。我们的跨学科 方法合并合成生物学，化学生物学，声学和固体力学，如果成功， 高风险的高增益项目将导致新一代的超敏感报道基因扩大 许多治疗和诊断应用的技术，尤其是在基于细胞的功能跟踪中 生物标志物的疗法和靶向成像。