Ren Shaokang, Fraser Keith, Kuo Lili, Chauhan Neha, Adrian Addison T, Zhang Fuming, Linhardt Robert J, Kwon Paul S, Wang Xing
Nick Holonyak Jr. Micro and Nanotechnology Laboratory (HMNTL), University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Nat Protoc. 2022 Feb;17(2):282-326. doi: 10.1038/s41596-021-00641-y. Epub 2022 Jan 10.
Emerging viral diseases can substantially threaten national and global public health. Central to our ability to successfully tackle these diseases is the need to quickly detect the causative virus and neutralize it efficiently. Here we present the rational design of DNA nanostructures to inhibit dengue virus infection. The designer DNA nanostructure (DDN) can bind to complementary epitopes on antigens dispersed across the surface of a viral particle. Since these antigens are arranged in a defined geometric pattern that is unique to each virus, the structure of the DDN is designed to mirror the spatial arrangement of antigens on the viral particle, providing very high viral binding avidity. We describe how available structural data can be used to identify unique spatial patterns of antigens on the surface of a viral particle. We then present a procedure for synthesizing DDNs using a combination of in silico design principles, self-assembly, and characterization using gel electrophoresis, atomic force microscopy and surface plasmon resonance spectroscopy. Finally, we evaluate the efficacy of a DDN in inhibiting dengue virus infection via plaque-forming assays. We expect this protocol to take 2-3 d to complete virus antigen pattern identification from existing cryogenic electron microscopy data, ~2 weeks for DDN design, synthesis, and virus binding characterization, and ~2 weeks for DDN cytotoxicity and antiviral efficacy assays.
新发病毒性疾病会对国家和全球公共卫生构成重大威胁。我们成功应对这些疾病的能力的核心在于需要快速检测出致病病毒并有效地将其中和。在此，我们展示了用于抑制登革病毒感染的DNA纳米结构的合理设计。设计的DNA纳米结构（DDN）可与分散在病毒颗粒表面的抗原上的互补表位结合。由于这些抗原以每种病毒特有的特定几何模式排列，DDN的结构被设计成与病毒颗粒上抗原的空间排列相匹配，从而提供非常高的病毒结合亲和力。我们描述了如何利用现有的结构数据来识别病毒颗粒表面抗原的独特空间模式。然后，我们介绍了一种使用计算机辅助设计原理、自组装以及凝胶电泳、原子力显微镜和表面等离子体共振光谱表征相结合的方法来合成DDN。最后，我们通过蚀斑形成试验评估了DDN抑制登革病毒感染的功效。我们预计，从现有的低温电子显微镜数据完成病毒抗原模式识别需要2至3天，DDN的设计、合成和病毒结合表征需要约2周时间，而DDN的细胞毒性和抗病毒功效检测需要约2周时间。
