Design of synthetic RNase-resistant RNA structures and their integration in novel riboswitch constructs to regulate mRNA stability

黄病毒是单链RNA病毒，其基因组RNA两侧是高度结构化的非翻译区（UTR）。在3 '-UTR内，进化上保守的RNA元件，称为外切核糖核酸酶抗性RNA（xrRNA），能够保护下游区域免受Xrn 1和相关5'-3 '-外切核酸酶的核酸外切降解，导致产生稳定的衰变中间体，其在感染细胞中积累为长非编码RNA（lncRNA）物质。这些病毒lncRNA，也称为亚基因组黄病毒RNA（sfRNA），介导这些病毒的致病性。我们将采用生物信息学方法来更好地了解xrRNA的2D和3D结构，这将使我们能够设计具有不同能力的合成xrRNA元件，以防止核酸外切降解。将通过将设计的元件整合到报告mRNA的5 '-UTR中来在体内和体外研究它们的核酸外切酶停滞能力，所述报告mRNA的开放阅读框（ORF）基于内部核糖体进入位点（IRES）进行翻译。这种保护的效率将通过确定mRNA半衰期来评估。此外，我们将利用设计的xrRNA获得的知识将RNA适体结构域与相容的xrRNA结构整合。通过将xrRNA与适体结合，我们将设计出核糖开关，其可以响应于特定配体的存在（或不存在）而形成保护结构。这些构建体将允许我们在配体络合时稳定（ON开关）或不稳定（OFF开关）特定mRNA。通过使用表现出不同能力的xrRNA元件来阻止核糖核酸外切酶，这将使我们能够修改单个转录本的半衰期。这种对RNA稳定性的诱导性调节代表了合成生物学中的一种新方法，该方法允许对mRNA和lncRNA进行精细调节，其半衰期由Xrn 1样核糖核酸外切酶决定。

Flaviviruses are single-stranded RNA viruses, whose genomic RNA is flanked by heavily structured untranslated regions (UTRs). Within the 3’-UTR, evolutionarily conserved RNA elements, referred to as exoribonuclease-resistant RNAs (xrRNAs), are capable of protecting downstream regions from exonucleolytic degradation by Xrn1 and related 5’-3’-exonucleases, resulting in the production of stable decay intermediates that accumulate as long non-coding RNA (lncRNA) species in infected cells. These viral lncRNAs, also termed subgenomic flavivirus RNAs (sfRNAs), mediate pathogenicity of these viruses. We will employ bioinformatics methods to better understand the 2D and 3D structure of xrRNAs, which will enable us to design synthetic xrRNA elements with varying capacity to protect against exonucleolytic degradation. Designed elements will be studied in vivo and in vitro for their exonuclease-stalling capacity by integrating them in the 5’-UTR of reporter mRNAs whose open reading frame (ORF) is translated based on an internal ribosome entry site (IRES). The efficiency of this kind of protection will be assessed by determining mRNA half-lives. Moreover, we will employ knowledge acquired with designed xrRNAs to integrate RNA aptamer domains with compatible xrRNA structures. By combining xrRNAs with aptamers we will devise riboswitches that can form a protective structure in response to presence (or absence) of a specific ligand. These constructs will allow us to stabilize (ON switch) or destabilize (OFF switch) a specific mRNA upon ligand complexation. By using xrRNA elements that exhibit different capacities to stall exoribonucleases, this will enable us to modify the half-lives of individual transcripts. This kind of inducible regulation of RNA stability represents a novel approach in synthetic biology that allows for fine-grained modulation of both mRNAs and lncRNAs, whose half-lives are determined by Xrn1-like exoribonucleases.