Programming designer DNA nanostructures for blocking enveloped viral infection

项目总结/摘要 SARS-CoV-2是COVID-19的病原体，已导致大流行。仍然迫切 需要创新技术，以促进开发负担得起的抗病毒精准医学。 SARS-CoV-2是一种有包膜的病毒，病毒粒子上的三聚体刺突蛋白簇的结构已经被证实是 解决了开发创新的、可负担的、生物相容的抗SARS-CoV-2病毒候选药物 和传播，我们利用病毒表面蛋白的结构特征，可以匹配在 通过工程DNA纳米结构平台实现纳米级精度。根据SARS-CoV-2的结构， 病毒体和表面刺突三聚体布局，我们已经合成了一种设计师DNA纳米结构（DDN）， 大分子“网”的形式，其顶点与 刺突蛋白基质展示在病毒外表面。我们假设，结构特性和 SARS-CoV-2刺突蛋白的布局模式可用于设计纳米级精度的DDN 其能够匹配和捕获具有结合SARS-CoV-2的亲合力的完整SARS-CoV-2病毒体， 选择性，从而阻断SARS-CoV-2感染。我们已经筛选并发现了DNA适体， 特异性针对刺突受体结合结构域（RBD）的纳米抗体。这些穗粘合剂可以是 结合到DDN网的“结”中，以允许多个DNA适体与 病毒表面的多个刺突以多价模式匹配的方式。DNA“网”-适体原型 构建体提供了SARS-CoV-2结合亲合力的显著增加。这个结构可以作为诱饵， 阻断病毒进入宿主细胞，比游离适体的效力高约1,000倍。在R21提案中， 我们的目标是扩展这项技术，使多种类型的探针对尖峰RBD的掺入， 验证DDNs在体外和体内抗病毒治疗中的安全性和有效性。我们提出两个具体的 目的：（1）设计、合成、验证并进一步优化针对各种SARS病毒的病毒捕获亲合力- 关注的CoV-2变体（VOC）;和（2）确定所设计的化合物的抗病毒效力和细胞毒性。 SARS-CoV-2体外感染人肺上皮细胞和体内ACE 2敲敲入过程中DDNs的变化 小鼠这项工作的完成将帮助我们确定DNA纳米结构的抗病毒效力和安全性， 被设计为完美匹配病毒表面上的表位布局以捕获和包裹活病毒。的 DDN治疗的估计成本约为10美元/剂（价格可能会大规模下降 合成），使其成为负担得起的治疗。这个DDN平台可能会进一步促进快速发展 针对新出现的SARS-CoV-2 VOC以及其他包膜病毒的抗病毒精准医学， 如流感和艾滋病。

Project Summary/Abstract SARS-CoV-2, the etiological pathogen of COVID-19, has resulted in a pandemic. There remains an urgent need for innovative technologies which facilitate the development of affordable antiviral precision medicine. SARS-CoV-2 is an enveloped virus, and the structure of the trimeric spike protein clusters on the virion has been solved. To develop innovative, affordable, and biocompatible antiviral candidates against SARS-CoV-2 infection and transmission, we exploited the structural characteristics of viral surface proteins that can be matched at nanoscale precision by engineered DNA nanostructure platforms. Based on the structure of the SARS-CoV-2 virion and surface spike trimer layout, we have synthesized a designer DNA nanostructure (DDN) that takes the form of a macromolecular ‘net’ whose vertices are a precise mechanical match to the spacing and positioning of the spike protein matrix displayed on the virus outer surface. We hypothesize that the structural properties and the layout patterns of SARS-CoV-2 spike proteins can be exploited to design DDNs with nanoscale precision which are capable of matching and capturing intact SARS-CoV-2 virions with ultrahigh binding avidity and selectivity, thereby blocking SARS-CoV-2 infection. We have screened and found DNA aptamers and nanobodies that are specific for the spike receptor-binding domain (RBD). These spike binders can be incorporated into the ‘knots’ of the DDN net to allow the simultaneous binding of multiple DNA aptamers with multiple spikes on the viral surface in a polyvalent, pattern-matching fashion. The DNA ‘net’-aptamer prototype construct has afforded dramatic increase in SARS-CoV-2 binding avidity. This construct can work as a decoy to block viral entry into host cells and is about 1,000-fold more potent than the free aptamer. In this R21 proposal, we aim to extend this technology to enable the incorporation of multiple types of probes against spike RBD and to validate the safety and effectiveness of DDNs in antiviral therapy in vitro and in vivo. We propose two specific aims: to (1) design, synthesize, validate, and further optimize the virus-capturing avidity against various SARS- CoV-2 variants of concern (VOCs); and (2) to determine the antiviral potency and cytotoxicity of the designed DDNs during SARS-CoV-2 infections in vitro in human lung epithelial cells and in vivo in human ACE2-knockin mice. Completion of this work will help us define the antiviral potency and safety of the DNA nanostructures that are designed to perfectly match epitope layouts on the viral surface to capture and wrap live viruses. The estimated cost of DDN treatment is approximately $10/dose (a price that likely decreases at large-scale synthesis), making it an affordable therapy. This DDN platform may further contribute to the rapid development of antiviral precision medicine against emerging SARS-CoV-2 VOCs, as well as other enveloped viruses such as influenza and HIV.