利用光鉗技術探討RNA偽結結構對核醣體框架位移之影響

Abstract

嚴重急性呼吸道症候群冠狀病毒SARS-CoV-1與SARS-CoV-2轉譯機制中的-1計畫性核醣體框架位移（-1 PRF）對於病毒複製與基因表達至關重要，其中促進-1 PRF發生要件除了由7個核苷酸所組成的滑動序列外，必須存在一個穩定的偽結結構作為移位刺激元件。為了抑制SARS-CoV-1與SARS-CoV-2活性，探討偽結摺疊過程與刺激框架位移的關聯性，已成為一個研究重點。 近年來有研究指出，一種稱為Merafloxacin的氟喹諾酮類抗菌化合物，對於β冠狀病毒有強烈抑制-1 PRF的特異性，由於β冠狀病毒的一個共同特徵是具備由三個莖（stems，依序稱為stem1，stem2，和stem3）摺疊而成偽結結構，因此推論Merafloxacin抑制-1 PRF的作用機制與偽結有關。針對此論點，我們將一系列相關藥物分別與SARS-CoV-2偽結混合，並透過單分子光鉗技術觀察偽結摺疊比例與解旋所需外力是否受到藥物影響而有所變化，藉此解釋藥物抑制-1 PRF效率與偽結的相關性。 mRNA偽結在轉譯過程中會被核醣體解旋，接著其序列由5’端往3’端依序從核醣體中釋放而開始摺疊。為了模擬真實轉譯狀態，我們修改了SARS-CoV-2偽結的設計，將序列拆成兩組RNA （CoV2-HP+CoV2-ST3n）再黏合，不僅能藉由CoV2-HP+CoV2-ST3n還原偽結構形，也能模擬其摺疊過程以及核醣體解旋的方式（經由最上游的stem1，而不是整個偽結）。除此之外，我們也利用5’ DNA handle 的互補序列延伸進入SARS-CoV-1與CoV2-HP+CoV2-ST3n stem1中，模擬轉譯過程核醣體對stem1摺疊所造成的影響。由實驗結果，推論由於核醣體立體結構干擾最穩定的stem1前幾個核苷酸，導致次穩定的stem3先形成，接著stem1與stem2再摺疊，此時3個stems會相互作用，最終形成穩定的偽結構形。在此情況下，框架位移的效率會最佳化，因此病毒基因可以正常表現。

The translation mechanism of -1 programmed ribosomal frameshifting (-1 PRF) in Severe Acute Respiratory Syndrome CoronaVirus SARS-CoV-1 and SARS-CoV-2 is crucial for virus replication and gene expression. In addition to the 7-nucleotide slippery sequence, a stable pseudoknot structure must be present as a frameshift-stimulating element for -1 PRF to occur. Exploring the folding process of pseudoknots and its association with frameshifting has become a major focus of research in order to inhibit the activity of SARS-CoV-1 and SARS-CoV-2. In recent years, studies have indicated that a fluoroquinolone antimicrobial compound called Merafloxacin exhibits strong specificity in inhibiting -1 PRF in betacoronaviruses. It has been inferred that the mechanism of Merafloxacin's inhibition of -1 PRF is related to the pseudoknot, as betacoronaviruses share the common feature of having a pseudoknot structure formed by three stems (stem1, stem2, and stem3). To examine this argument, a series of drugs were added to the pseudoknot structure of SARS-CoV-2, and the proportions of various folded conformations and the unfolding force before and after drug addition were observed using single-molecule optical tweezers techniques. This was done to determine if the efficiency of -1 PRF inhibition by the drugs is related to the pseudoknot. mRNA pseudoknots are unfolded by ribosomes during translation, and then refold in a vectorial manner from the 5’ to the 3’ ends when the strand is gradually released from the ribosome. To mimic the actual translation state, the design of the SARS-CoV-2 construct was modified by splitting the sequence into two sets of single-stranded RNA (ssRNA), which were then annealed to create CoV2-HP+CoV2-ST3n. This annealed construct not only allows the restoration of the complete pseudoknot conformation, but also mimic the ribosome-meidated unfolding (through stem1, instead of the whole pseudoknot) and refolding (the 5’-to-3’ direction) pathways. Additionally, we also extended the 5’handle into stem1 of SARS-CoV-1 and CoV2-HP+CoV2-ST3n to mimic the impact of ribosome-induced folding on stem1 during the translation process. From the experimental results, we hypothesize that, owing to the ribosome’s (or handle’s) interference on the first few nucleotides of stem1, stem2 will take turn to form first, followed by stem1 and stem3. This folding pathway will result in a highly stable pseudoknot, including the further strengthened stem1. Such ribosome-mediated pseudoknot refolding will in turn result in an optimal frameshifting efficiency to facilitate viral gene expression and replication.