Kazakov Sergei A, Balatskaya Svetlana V, Johnston Brian H
Somagenics, Inc., 2161 Delaware Avenue, Santa Cruz, CA 95060, USA.
RNA. 2006 Mar;12(3):446-56. doi: 10.1261/rna.2123506.
Although reducing the temperature slows most chemical reactions, freezing can stimulate some reactions by mechanisms that are only partially understood. Here we show that freezing stimulates the self-ligation (circularization) of linear forms of the hairpin ribozyme (HPR) containing 2',3'-cyclic phosphate and 5'-OH termini. Divalent metal ions (M2+) are not required, but monovalent cations and anions at millimolar concentrations can have various effects on this reaction depending on the specific ion. Under optimal conditions, the observed rate of M2+-independent self-ligation reaches a peak (0.04 min(-1)) at -10 degrees C with a yield of -60% after 1 h. In contrast, no ligation occurs either at above 0 degrees C or in solutions that remain unfrozen when supercooled to subzero temperatures. Under freezing conditions, the cleavage-ligation equilibrium strongly favors ligation. Besides freezing, evaporation of the aqueous solvent as well as the presence of ethanol at levels of 40% or above can also induce M2+-independent HPR ligation at 25 degrees C. We argue that partial RNA dehydration, which is a common feature of freezing, evaporation, and the presence of ethanol, is a key factor supporting HPR ligation activity at both above- and below-freezing temperatures. In the context of the RNA world hypothesis, freezing-induced ligation is an attractive mechanism by which complex RNAs could have evolved under conditions in which RNA was relatively protected against degradation.
尽管降低温度会减缓大多数化学反应，但冷冻却能通过一些仅被部分理解的机制刺激某些反应。在此我们表明，冷冻能刺激含有2',3'-环磷酸和5'-羟基末端的发夹状核酶（HPR）线性形式的自我连接（环化）。二价金属离子（M2+）并非必需，但毫摩尔浓度的单价阳离子和阴离子会根据具体离子对此反应产生不同影响。在最佳条件下，观察到的不依赖M2+的自我连接速率在-10℃时达到峰值（0.04 min-1），1小时后的产率为-60%。相比之下，在0℃以上或过冷至零下温度仍未冻结的溶液中均不会发生连接。在冷冻条件下，切割-连接平衡强烈倾向于连接。除了冷冻外，水性溶剂的蒸发以及40%或更高浓度乙醇的存在也能在25℃诱导不依赖M2+的HPR连接。我们认为，部分RNA脱水是冷冻、蒸发和乙醇存在的共同特征，是在高于和低于冰点温度下支持HPR连接活性的关键因素。在RNA世界假说的背景下，冷冻诱导的连接是一种有吸引力的机制，通过该机制复杂的RNA可能在RNA相对受到保护不被降解的条件下进化而来。
