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: 10684322
• 负责人: George J Lu
• 金额: $ 18.99万
• 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/10684322
• 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; cellular imaging; crosslink; design; disease diagnosis; fluorescence imaging; hemodynamics; high risk; imaging agent; imaging biomarker; improved; in vitro testing; innovation; interdisciplinary approach; mechanical properties; monomer; new technology; non-invasive imaging; particle; pressure; screening; simulation; submicron; synthetic biology; targeted imaging; technology development; 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.

项目总结/摘要 对特定基因表达和细胞信号传导途径成像的能力长期以来一直是一种强有力的工具， 科学家，以及实现这一目标的方法，如荧光报告基因，被广泛使用， 如今在不同的生物医学领域。然而，光的穿透深度限制了 荧光报告基因在动物模型和基于细胞的疗法在人类。为此目的。一组气体- 最近引入了填充的蛋白质纳米结构（称为气泡（GVs））作为声学报告基因 并首次实现了使用超声成像来可视化细胞中特定基因的表达， 一厘米深的组织GV是亚微米颗粒，最初在光合微生物中进化， 细胞漂浮，并且，这些野生型GV不具有可以最大化 通过超声波检测它们。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。在本建议中，我们的目标是引入第二个- 通过创新蛋白质壳的机械性质来产生声学报告基因。在目标1中，我们 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，蛋白质外壳的向内弯曲是敏感的关键。 这些声学蛋白质纳米结构的检测，但往往导致野生型GV壳的破裂， 系统交联将显著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， 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平行，我们将在目标2中开发有限元建模和声学测量方法， 了解交联对壳体机械性能的影响，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的声学蛋白纳米结构。我们的跨学科 这种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， 高风险高收益项目将导致新一代超灵敏的声音报告基因， 该技术可用于许多治疗和诊断应用，特别是在基于细胞的功能跟踪中。 生物标记物的靶向成像。