Abstract：
SARS-CoV-2 is a betacoronavirus that causes COVID-19, a global pandemic that has resulted in many infections, deaths, and socio-economic challenges. The virus has a large positive-sense, single-stranded RNA genome of ∼30 kb, which produces subgenomic RNAs (sgRNAs) through discontinuous transcription. The most abundant sgRNA is sgRNA N, which encodes the nucleocapsid (N) protein. In this study, we probed the secondary structure of sgRNA N and a shorter model without a 3\' UTR in vitro, using the SHAPE (selective 2\'-hydroxyl acylation analyzed by a primer extension) method and chemical mapping with dimethyl sulfate and 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate. We revealed the secondary structure of sgRNA N and its shorter variant for the first time and compared them with the genomic RNA N structure. Based on the structural information, we designed gapmers, siRNAs and antisense oligonucleotides (ASOs) to target the N protein coding region of sgRNA N. We also generated eukaryotic expression vectors containing the complete sequence of sgRNA N and used them to screen for new SARS-CoV-2 gene N expression inhibitors. Our study provides novel insights into the structure and function of sgRNA N and potential therapeutic tools against SARS-CoV-2.
摘要：
SARS-CoV-2是一种导致COVID-19的β冠状病毒，这是一种导致许多感染的全球大流行，死亡,和社会经济挑战。这种病毒有很大的积极意义，30kb的单链RNA基因组，通过不连续转录产生亚基因组RNA（sgRNA）。最丰富的sgRNA是sgRNAN,其编码核衣壳(N)蛋白。在这项研究中,我们在体外探测了sgRNAN的二级结构和没有3'UTR的较短模型，使用SHAPE（通过引物延伸分析的选择性2'-羟基酰化）方法和硫酸二甲酯和1-环己基-（2-吗啉代乙基）碳二亚胺甲基-对甲苯磺酸盐的化学作图。我们首次揭示了sgRNAN及其较短变体的二级结构，并将它们与基因组RNAN结构进行了比较。根据结构信息，我们设计了缺口器，siRNA和反义寡核苷酸（ASO）靶向sgRNAN的N蛋白编码区。我们还生成了包含sgRNAN完整序列的真核表达载体，并将其用于筛选新的SARS-CoV-2基因N表达抑制剂。我们的研究为sgRNAN的结构和功能以及针对SARS-CoV-2的潜在治疗工具提供了新的见解。
