![]() ![]() The DNA star was designed to afford local structural flexibility through a single-stranded DNA (ssDNA) region on each of the five internal edges and unpaired thymine bases (Ts) at the five internal junctions. ![]() We anticipate that our strategy can be tailored to target any viral epitope pattern to combat emerging and re-emerging viruses by generating the requisite ligand patterns on customized designer DNA nanostructures (DDNs). The DNA star demonstrated high virus-binding avidity and specificity to DENV and was a highly potent DENV inhibitor in human blood with a half-maximal effective concentration (EC 50) of 2 nM (~7,500-fold more effective than the monovalent aptamer). Each DENV (~50 nm diameter) can bind up to two DNA stars, one on each hemisphere. The five-point DNA star provides structural rigidity to display ten dengue ED3 targeting aptamers in a 2D pattern precisely mirroring the complex spatial arrangement of DENV ED3s 7. We designed the antiviral DNA star to specifically target complex epitopes on the DENV envelope protein domain 3 (ED3). Our group recently designed and synthesized a ~43 nm diameter star-shaped DNA architecture, called a ‘DNA star’ to multivalently bind to viral epitopes to efficiently inhibit virus infection. Development of the DNA star antiviral platform Based on this naturally occurring multivalent virus-cell binding mechanism, creating polyvalent virus entry blockers is a promising and practical approach to producing potent inhibitors of virus infections. Such patterns facilitate multivalent binding of the virus to host cells for enhanced pathogenic infectivity. Viruses present unique spatial patterns of antigens on their surfaces 6. Importantly, NAbs may induce unwanted antibody-dependent enhancement of infection 4, 5 (for example, with dengue virus (DENV) vaccine), where antibodies induce increased viral infectivity in vivo. However, producing antibodies for treatment is very costly and time consuming. Therapeutic antibodies can be administered in response to viral infections. However, safe and effective vaccines normally take years to develop for an emerging virus. Production of NAbs can be triggered by vaccination or active virus infection in the host. Inhibition and treatment of virus infections typically relies on neutralizing antibodies (NAbs) that target virus surface-specific epitopes mainly in a one-to-one fashion 3. The challenges underlying current coronavirus disease 2019 treatment and rapid diagnostic development are already well known from previous encounters with newly emerging pathogens (e.g., the 2009 H1N1 pandemic 1, 2). 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. Finally, we evaluate the efficacy of a DDN in inhibiting dengue virus infection via plaque-forming assays. 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. We describe how available structural data can be used to identify unique spatial patterns of antigens on 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. The designer DNA nanostructure (DDN) can bind to complementary epitopes on antigens dispersed across the surface of a viral particle. Here we present the rational design of DNA nanostructures to inhibit dengue virus infection. Central to our ability to successfully tackle these diseases is the need to quickly detect the causative virus and neutralize it efficiently. ![]() Emerging viral diseases can substantially threaten national and global public health. ![]()
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