Green Michael R, Sambrook Joseph
Cold Spring Harb Protoc. 2019 Feb 1;2019(2):2019/2/pdb.top101857. doi: 10.1101/pdb.top101857.
Because ribose residues carry hydroxyl groups in both the 2' and 3' positions, RNA is chemically much more reactive than DNA and is easy prey to cleavage by contaminating RNases-enzymes with various specificities that share the property of hydrolyzing diester bonds linking phosphate and ribose residues. Because RNases are released from cells following lysis and are present on the skin, constant vigilance is required to prevent contamination of glassware and bench tops and the creation of aerosols carrying RNase. The problem is compounded because there is no simple method to inactivate RNases. Because of the presence of intrachain disulfide bonds, many RNases are resistant to prolonged boiling and mild denaturants and are able to refold quickly when denatured. Unlike many DNases, RNases do not require divalent cations for activity and thus cannot be easily inactivated by the inclusion of ethylenediaminetetraacetic acid (EDTA) or other metal ion chelators in buffer solutions. The best way to prevent problems with RNase is to avoid contamination in the first place.
由于核糖残基在2'和3'位置都带有羟基,RNA在化学性质上比DNA更具反应性,很容易被污染的核糖核酸酶切割——核糖核酸酶是一类具有各种特异性的酶,它们都具有水解连接磷酸和核糖残基的二酯键的特性。由于核糖核酸酶在细胞裂解后会从细胞中释放出来,并且存在于皮肤上,因此需要时刻保持警惕,以防止玻璃器皿和实验台面受到污染,以及防止产生携带核糖核酸酶的气溶胶。问题因没有简单的方法使核糖核酸酶失活而变得更加复杂。由于存在链内二硫键,许多核糖核酸酶对长时间煮沸和温和变性剂具有抗性,并且在变性后能够迅速重新折叠。与许多脱氧核糖核酸酶不同,核糖核酸酶的活性不需要二价阳离子,因此在缓冲溶液中加入乙二胺四乙酸(EDTA)或其他金属离子螯合剂并不能轻易使其失活。防止核糖核酸酶问题的最佳方法是首先避免污染。