Mortimer Stefanie A, Weeks Kevin M
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA.
Nat Protoc. 2009;4(10):1413-21. doi: 10.1038/nprot.2009.126. Epub 2009 Sep 10.
RNA selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt configurations that facilitate reaction between the 2'-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2'-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and ribonucleoprotein (RNP) assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 degrees C. RNA folding is initiated by addition of Mg(2+) or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then 'waiting' for 1 second. Sites of the 2'-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time-resolved SHAPE protocol makes it possible to obtain 1-second structural snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment.
通过引物延伸分析RNA选择性2'-羟基酰化（SHAPE）化学方法利用了这样一个发现：构象动态的核苷酸优先采用有助于2'-OH基团与羟基选择性亲电试剂（如苯甲酰氰（BzCN））之间反应的构型，以形成2'-O-加合物。BzCN非常适合用于RNA折叠和核糖核蛋白（RNP）组装机制的定量、时间分辨分析，因为该试剂既能与柔性RNA核苷酸反应，又能在37℃下以0.25秒的半衰期进行自动失活水解。通过添加Mg(2+)或蛋白质或溶液条件的其他变化启动RNA折叠，通过将不断演变的反应等分试样添加到BzCN中，然后“等待”1秒来获得核苷酸分辨率的结构图像。随后，使用逆转录酶将2'-O-加合物形成的位点计为引物延伸的终止点。这种时间分辨的SHAPE方案使得在一个直接而简洁的实验中，能够在时间分辨动力学研究中为任意长度和复杂性的RNA获得1秒的结构快照。
