Department of Infectious Diseases, Renmin Hospital, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, China.
Institute of Virology, Virology Key Laboratory of Shiyan City, Shiyan, China.
Microbiol Spectr. 2024 Apr 2;12(4):e0341023. doi: 10.1128/spectrum.03410-23. Epub 2024 Feb 20.
The nucleocapsid protein of SARS-CoV-2 plays significant roles in viral assembly, immune evasion, and viral stability. Due to its immunogenicity, high expression levels during COVID-19, and conservation across viral strains, it represents an attractive target for antiviral treatment. In this study, we identified and characterized a single-stranded DNA aptamer, N-Apt17, which effectively disrupts the liquid-liquid phase separation (LLPS) mediated by the N protein. To enhance the aptamer's stability, a circular bivalent form, cb-N-Apt17, was designed and evaluated. Our findings demonstrated that cb-N-Apt17 exhibited improved stability, enhanced binding affinity, and superior inhibition of N protein LLPS; thus, it has the potential inhibition ability on viral replication. These results provide valuable evidence supporting the potential of cb-N-Apt17 as a promising candidate for the development of antiviral therapies against COVID-19.IMPORTANCEVariants of SARS-CoV-2 pose a significant challenge to currently available COVID-19 vaccines and therapies due to the rapid epitope changes observed in the viral spike protein. However, the nucleocapsid (N) protein of SARS-CoV-2, a highly conserved structural protein, offers promising potential as a target for inhibiting viral replication. The N protein forms complexes with genomic RNA, interacts with other viral structural proteins during virion assembly, and plays a critical role in evading host innate immunity by impairing interferon production during viral infection. In this investigation, we discovered a single-stranded DNA aptamer, designated as N-Apt17, exhibiting remarkable affinity and specificity for the N protein. Notably, N-Apt17 disrupts the liquid-liquid phase separation (LLPS) of the N protein. To enhance the stability and molecular recognition capabilities of N-Apt17, we designed a circular bivalent DNA aptamer termed cb-N-Apt17. In both and experiments, cb-N-Apt17 exhibited increased stability, enhanced binding affinity, and superior LLPS disrupting ability. Thus, our study provides essential proof-of-principle evidence supporting the further development of cb-N-Apt17 as a therapeutic candidate for COVID-19.
新型冠状病毒的核衣壳蛋白在病毒组装、免疫逃逸和病毒稳定性方面发挥着重要作用。由于其免疫原性、在 COVID-19 期间的高表达水平以及在病毒株之间的保守性，它是抗病毒治疗的一个有吸引力的靶点。在这项研究中，我们鉴定并表征了一种单链 DNA 适体 N-Apt17，它可以有效地破坏 N 蛋白介导的液-液相分离（LLPS）。为了提高适体的稳定性，设计并评估了一种环状二价形式 cb-N-Apt17。我们的研究结果表明，cb-N-Apt17 表现出了更好的稳定性、增强的结合亲和力和对 N 蛋白 LLPS 的更好抑制作用；因此，它具有抑制病毒复制的潜力。这些结果为 cb-N-Apt17 作为一种有前途的 COVID-19 抗病毒治疗候选物提供了有价值的证据。
新型冠状病毒的变异株对目前可用的 COVID-19 疫苗和疗法构成了重大挑战，这是因为在病毒刺突蛋白中观察到了快速的表位变化。然而，新型冠状病毒的核衣壳（N）蛋白是一种高度保守的结构蛋白，作为抑制病毒复制的靶标具有很大的潜力。N 蛋白与基因组 RNA 形成复合物，在病毒组装过程中与其他病毒结构蛋白相互作用，并通过在病毒感染过程中损害干扰素的产生来逃避宿主先天免疫。在这项研究中，我们发现了一种单链 DNA 适体，命名为 N-Apt17，它对 N 蛋白具有显著的亲和力和特异性。值得注意的是，N-Apt17 破坏了 N 蛋白的液-液相分离（LLPS）。为了提高 N-Apt17 的稳定性和分子识别能力，我们设计了一种环状二价 DNA 适体 cb-N-Apt17。在 和 实验中，cb-N-Apt17 表现出了更高的稳定性、增强的结合亲和力和更好的 LLPS 破坏能力。因此，我们的研究为进一步将 cb-N-Apt17 开发为 COVID-19 的治疗候选物提供了重要的原理验证证据。
