Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group
Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2′-hydroxyl group
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DOI：
10.1021/ja990592p
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发表时间：
1999-06-16
影响因子：
15
通讯作者：
Breaker, RR
中科院分区：
文献类型：
--
作者：
Li, YF;Breaker, RR
详细了解RNA磷酸二酯对特异性碱催化裂解的敏感性对于在各种条件下近似RNA的稳定性是必要的。此外，需要定量的速率增强，可以专门由这种常见的切割机制，以充分解释核糖核酸酶和RNA切割核酶所采用的机制。嵌合的DNA/ RNA寡核苷酸被用来检查在模拟生物系统的反应条件下RNA磷酸二酯的氢氧化物依赖性降解的速率。在中性或碱性pH条件下，RNA降解的主要途径是由特定碱催化促进的内部磷酸酯转移反应。如所预期的，增加氢氧根离子的浓度、增加二价镁的浓度或升高温度加速链断裂。在大多数情况下，靶RNA连接侧翼的核苷酸碱基的身份对亲核2 '-羟基的pK（a）具有可忽略的影响，并且对酯交换反应的最大速率常数仅具有微小的影响。在代表性的生理条件下，RNA切割的特异性碱基催化产生的最大速率增强类似于100 000倍以上的RNA酯交换的背景速率。本文报道的动力学参数为RNA聚合物的稳定性和RNA切割酶和酶模拟物的熟练程度提供了理论极限，所述酶模拟物专门采用一般碱催化的机制。
A detailed understanding of the susceptibility of RNA phosphodiesters to specific base-catalyzed cleavage is necessary to approximate the stability of RNA under various conditions. In addition, quantifying the rate enhancements that can be produced exclusively by this common cleavage mechanism is needed to fully interpret the mechanisms employed by ribonucleases and by RNA-cleaving ribozymes. Chimeric DNA/ RNA oligonucleotides were used to examine the rates of hydroxide-dependent degradation of RNA phosphodiesters under reaction conditions that simulate those of biological systems. Under neutral or alkaline pH conditions, the dominant pathway for RNA degradation is an internal phosphoester transfer reaction that is promoted by specific base catalysis. As expected, increasing the concentration of hydroxide ion, increasing the concentration of divalent magnesium, or raising the temperature accelerates strand scission. In most instances, the identities of the nucleotide bases that flank the target RNA linkage have a negligible effect on the pK(a) of the nucleophilic 2'-hydroxyl group, and only have a minor effect on the maximum rate constant for the transesterification reaction. Under representative physiological conditions, specific base catalysis of RNA cleavage generates a maximum rate enhancement of similar to 100 000-fold over the background rate of RNA transesterification. The kinetic parameters reported herein provide theoretical limits for the stability of RNA polymers and for the proficiency of RNA-cleaving enzymes and enzyme mimics that exclusively employ a mechanism of general base catalysis.