Ultrasensitive acoustic reporter proteins with engineered rupture-resistant shells

项目摘要/摘要 长期以来，成像特定基因表达和细胞信号通路的能力一直是 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 如今跨越了生物医学的不同领域。然而，光的穿透深度限制了它的使用。 动物模型和人类细胞疗法中的荧光报告基因。为了这个目的。一群气体- 填充的蛋白质纳米结构称为气泡(GVS)，是最近引入的声学报告基因 并首次实现了使用超声波成像来可视化定位的细胞中的特定基因表达 在厘米深的组织中。GVS是一种亚微米颗粒，最初在光合作用微生物中进化，以实现 细胞漂浮，意想不到的是，这些野生型GV没有正确的属性，可以最大限度地 他们的超声波检测。这种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。在这项建议中，我们的目标是引入第二项- 通过创新蛋白质壳的机械性能来产生声学报告基因。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。这是要认识到蛋白质外壳的向内弯曲掌握着敏感的 检测到这些声学蛋白质纳米结构，但往往会导致野生型GV壳的破裂，并且 系统的交联会大大提高这些蛋白质壳的抗拉强度。具体来说，我们将 系统地搜索允许非规范氨基酸和赖氨酸结合的站点，以获得接近- 诱导化学，这项研究将在结构模型的合理指导下进行。在……里面 与目标1平行，我们将在目标2中发展有限元建模和声学测量方法，以 了解交联剂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断裂的声学蛋白纳米结构。我们的跨学科 该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，这 高风险高收益项目将导致新一代超敏感声学报告基因 这项技术应用于许多治疗和诊断，特别是在基于细胞的功能跟踪 生物标志物的治疗和靶向成像。

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.