De novo design of a generalizable protein biosensor platform for point-of-care testing

úú项目摘要/摘要 在医疗点(POC)提供诊断服务可以提高诊所的医疗质量 紧急情况下，或在家里，这可能会减轻医院的负担，例如，在COVID期间- 19大流行。精确化和个性化的医学革命也需要POC测试来随时提供 可供临床医生使用的生物标志物信息。这份职业发展提案的目标是快速创造、 廉价、灵敏和可靠的分子诊断技术，以应对21世纪的医疗保健挑战。 中心假设是，我们可以有效地利用计算蛋白质设计来创建模块 变构蛋白开关，称为LOCKR(闩锁正交笼关键蛋白)，使快速 以及相互作用时的可逆构象变化。作为原则的证明，我们证明了LOCKR- 基于生物传感器的可被配置为在添加临床靶标时产生生物发光(例如， 肉毒毒素，心肌肌钙蛋白I，HER2受体，Fc结构域，抗HBVmAb，抗SARS-CoV2抗体， 和SARS-CoV2受体结合域/刺突蛋白，图1和图2)均一的“All-in-Solution”中 化验。由于LOCKR传感器平台的模块化和从头开始的粘合剂设计的进步 对于任意的蛋白质靶点，我们提出了将这两种特征相结合作为通用的开发策略 为用户定义的目标定制的生物传感器。独立阶段的主要具体目标是 迭代地扩展基于LOCKR的诊断，协同(1)从头开始的蛋白质结合设计，以直接 检测各种疾病蛋白生物标志物，以及(2)间接检测与 设计接口，作为PoC设备；以及(3)将原始发光信号与其他 通过交换报告器模块来兼容读数。对于更具体的概念验证项目，在 指导阶段，我在目标1中描述了使用最先进的计算蛋白质设计方法来 制作一种白介素6粘合剂和生物传感器。在目标2中，我提出了一种制备抗体的一般方法 以新冠肺炎的血清学试验为例进行生物传感器的演示。以我在生物传感器方面的专业知识 在工程方面，我尝试在目标3中开发一种比率生物发光共振能量转移(Bret) 用于分析乙肝病毒抗体的生物传感器和用于检测人心肌肌钙蛋白I的比色生物传感器 水平。最终，我预计这个新的传感器平台对强健蛋白质的开发具有重要意义 传感器将广泛适用于任意目标，并为未来启用与POC兼容的读数 诊断。

úú PROJECT SUMMARY/ABSTRACT Delivering diagnostic services at the point-of-care (POC) can improve the quality of healthcare in clinics, in emergency settings, or at home, which can potentially ease hospitals’ burden, for instance, during the COVID- 19 pandemic. Precision and personalized medicine revolution also require POC testing to provide readily available biomarker information to clinicians. The goal of this career development proposal is to create fast, inexpensive, sensitive, and reliable molecular diagnostics to address the 21st-century healthcare challenges. The central hypothesis is that we can efficiently utilize computational protein design to create modular allosteric protein switches, named LOCKR (Latching Orthogonal Cage–Key pRotein), that enable the rapid and reversible conformational changes upon interaction. As a proof of principle, we demonstrate that LOCKR- based biosensors can be configured to produce bioluminescence upon the addition of clinical targets (e.g., botulinum toxin, cardiac troponin I, HER2 receptor, Fc domain, anti-HBV mAb, anti-SARS-CoV2 antibodies, and SARS-CoV2 receptor-binding domain/spike protein, Fig 1 and 2) in homogeneous “all-in-solution” assays. Due to the modularity of LOCKR sensor platform and the advance in de novo binder design for arbitrary protein targets, we proposed the integration of both features as the universal strategy to develop tailored biosensors for user-defined targets. The main specific aims for the independent phase are to iteratively expand LOCKR-based diagnostics with the synergy of (1) de novo protein binder design to directly detect various disease protein biomarkers, and (2) indirectly detect the antibodies that compete with the designed interface, as POC devices; and (3) to repurpose the original luminescence signal with other compatible readouts by exchanging the reporter modules. For more specific proof-of-concept projects during the mentored phase, I describe in Aim 1 the use of state-of-the-art computational protein design methods to create an interleukin-6 binder and biosensor. In Aim 2, I propose a general way to develop antibody biosensors by demonstrating COVID-19 serological tests as an example. With my expertise in biosensor engineering, I attempt in Aim 3 to develop a ratiometric bioluminescence resonance energy transfer (BRET) biosensor to analyze the HBV antibody and a colorimetric biosensor to measure human cardiac troponin I level. Ultimately, I anticipate this new sensor platform is significant for the development of robust protein sensors that will be broadly applicable to arbitrary targets and enabling its POC compatible readouts for future diagnostics.