利用單分子螢光共振能量轉移技術探討SARS-CoV-2偽結的摺疊

Abstract

許多種類的病毒或細菌等擁有較短信使核醣核酸(mRNA)的物種會藉由核醣體重複讀取一個核苷酸，使下游核苷酸密碼子重新排列進而合成出不同的蛋白質，這種機制稱為-1框架位移，而-1框架位移發生的條件需要有滑動序列以及距離下游5-7個核苷酸的二級結構(例如偽結)。本次實驗我們使用SARS-CoV-2偽結RNA作為實驗材料，SARS-CoV-2為一RNA病毒並且為COVID-19的感染源，值得注意的是SARS-CoV-2偽結RNA具典型冠狀病毒RNA的三莖三環結構，並有著15-30%的-1框架位移效率。 我們透過與互補DNA的黏合解開SARS-CoV-2偽結的二級結構，再將互補DNA由3’端到5’端的逐步與RNA分離以模擬核醣體在mRNA轉譯的過程，並透過單分子螢光共振能量轉移 (smFRET)技術測量SARS-CoV-2偽結於再摺疊過程中特定兩點的距離變化，以推斷其構型的改變及探討中間態形成與否與其對-1框架位移效率的影響。我們分別使用DNA polymerase I, Klenow Fragment、Exonuclease V (RecBCD)、phi29 DNA polymerase進行RNA/DNA的分離，發現Klenow fragment無法成功將互補的DNA去除。而Exonuclease V能有效去除DNA並使SARS-CoV-2偽結重新摺疊，然而，由於Exonuclease V的作用是雙向並可能會造成RNA結構不穩定。而SARS-CoV-2 RNA 在phi29 DNA polymerase作用下能明顯觀察出RNA逐步形成偽結的過程，並能觀察到部份RNA再摺疊的過程中有中間態短暫的形成，這些中間態是否會影響-1框架位移效率目前尚不清楚，仍需要後續不同具框架位移機制的RNA互相比較。

Minus-one programmed ribosomal frameshifting (-1 PRF) is a translational mechanism in several types of viruses and bacteria. It occurs when the ribosome rereads a nucleotide and therefore changes the reading frame in order to express multiple proteins from a single mRNA. In order to stimulate -1 PRF, a slippery sequence and a well-spaced downstream RNA secondary structure, such as a pseudoknot, should be contained in mRNA. In this experiment, we used the frameshift-stimulating pseudoknot RNA derived from SARS-CoV-2, which is an RNA virus that causes COVID-19, as our model. It is noteworthy that the SARS-CoV-2 pseudoknot RNA is a traditional three stem-three loop pseudoknot structure and its –1 frameshift efficiency is about 15-30%. We used single-molecule Förster resonance energy transfer (smFRET) to observe the folding of SARS-CoV-2 pseudoknot RNA. Native and intermediate conformations may be formed when RNA refolds during ribosomal translation. To simplify the experiment and mimic the ribosomal translation, we annealed SARS-CoV-2 pseudoknot RNA with its complementary DNA and then the RNA was released from the 5’ to 3’ end gradually by removing the DNA strand. DNA polymerase I Klenow Fragment, Exonuclease V (RecBCD), and phi29 DNA polymerase were used to release RNA. We found that DNA polymerase I Klenow Fragment isn’t able to displace the RNA strand. Exonuclease V can digest the complementary DNA to release the pseudoknot RNA, but it acts bidirectionally, which can also digest from the opposite end of DNA. When treating with phi29 DNA polymerase, which has stronger strand displacement ability than the Klenow Fragment, transient intermediates can be observed during RNA refolding to pseudoknots. However, whether these intermediates influence -1 PRF is still unknown and should be compared to other frameshift-stimulating RNA in the future.