利用光鉗探討小分子藥物對於框架位移刺激子RNA偽結的效應

Abstract

當核醣體在轉譯過程中遇到mRNA上的滑動序列(slippery sequence)和下游刺激子(stimulator) RNA 偽結，會有一定機率往上游移動一個核苷酸而改變閱讀框架，稱為 -1計畫性核醣體框架位移(-1 programmed ribosomal frameshifting, -1 PRF)，造成後續轉譯出的蛋白質序列不同，-1 PRF對於合成生物學的應用及許多病毒基因表達至關重要。在眾多-1 PRF刺激子中，DU177偽結具有較高的效率與清楚的結構資訊，所以我們選用DU177作為模板，研究如何利用小分子藥物為配體來調控-1 PRF，而且前人的研究也成功利用DU177的突變體搭配特定的小分子配體來調控-1 PRF的效率，但其調控的分子機制並不清楚。在此論文中，我們運用相同的設計將DU177的stem突變成帶有5'-CGG-3'/3'-GGC-5'的序列而破壞其結構(-1 PRF效率下降)，但小分子藥物NCT8可與該區域的GG mismatch結合而回復結構穩定性(效率回升)，我們利用單分子光鉗技術探討NCT8與RNA結構的結合機制。實驗的結果發現NCT8對於stem 1上帶有5'-CGG-3'/3'-GGC-5'的construct有影響，藥物讓低穩定偽結回復到與原生型DU177相似的高穩定偽結，在對照組帶有5'-CGG-3'/3'-GGC-5'的髮夾結構中，藥物有增加結構穩定性的趨勢。 另一方面，一種稱為Merafloxacin的氟喹諾酮類抗菌化合物，對於SARS-CoV-2冠狀病毒有抑制其-1 PRF的特性，SARS-CoV-2的-1 PRF刺激子是由三個莖（依序稱為stem 1，stem 2，和stem 3）摺疊的偽結結構，因此推論藥物抑制-1 PRF的作用機制可能與這些二級結構的摺疊有關。我們透過光鉗觀察一系列相關小分子藥物對於SARS-CoV-2 RNA偽結摺疊過程的影響，探討藥物抑制病毒-1 PRF效率的機制，實驗結果顯示藥物對SARS-CoV-2偽結的stem 2和stem 3摺疊有影響，藥物與stem 2和stem 3結合，結構處於不穩定狀態無法摺疊成穩定偽結，及改變構形間轉換的比例，進而影響到-1 PRF的效率。

During the translation process, when the ribosome encounters the slippery sequence and downstream stimulator RNA pseudoknot on the mRNA, there is a probability of shifting one nucleotide upstream, altering the reading frame. This phenomenon is known as -1 programmed ribosomal frameshifting (-1 PRF), leading to a different protein sequence being translated. -1 PRF is crucial for synthetic biology applications and the gene expression of many viruses. Among numerous -1 PRF stimulators, the DU177 pseudoknot exhibits high efficiency and clear structural information, making it an ideal template for studying how small molecule ligands can modulate -1 PRF. Previous research successfully utilized mutants of DU177 along with specific small molecule ligands to modulate -1 PRF efficiency. However, the molecular mechanism underlying this regulation remains unclear. In this study, we designed DU177 mutants where the stem was partially disrupted by introducing a 5'-CGG-3'/3'-GGC-5' sequence, leading to decreased -1 PRF efficiency. The NCT8 ligands can target the GG mismatch motif, restoring structural stability and thereby restoring -1 PRF efficiency. Subsequently, we employed optical tweezers to investigate the binding mechanism between NCT8 and the RNA structure. Our data showed that NCT8 affects constructs containing the 5'-CGG-3'/3'-GGC-5' sequence on stem 1, restoring low-stability pseudoknots to a high-stability state similar to the native DU177. Additionally, in control hairpin structures with the 5'-CGG-3'/3'-GGC-5' sequence, the drug showed a trend of increased structural stability. On the other hand, a fluoroquinolone antibiotic compound called Merafloxacin exhibits properties of inhibiting -1 PRF in SARS-CoV-2. The -1 PRF stimulator of SARS-CoV-2 a pseudoknot formed by three stems (termed stem 1, stem 2, and stem 3), suggesting that the mechanism of action of Merafloxacin in inhibiting -1 PRF may be related to the folding of these secondary structures. We investigated a series of related small-molecule drugs using optical tweezers to observe their effects on the folding of SARS-CoV-2 RNA pseudoknots, aiming to elucidate the mechanism by which these drugs inhibit viral -1 PRF efficiency. Our data showed that the drugs affect the folding of stem 2 and stem 3 of the SARS-CoV-2 pseudoknot structure. By binding to stem 2 and stem 3, the drugs destabilize their structures and prevente them from folding into a highly stable pseudoknot. This alteration affects the ratio of conformational transitions, thereby influencing the efficiency of -1 PRF.