A DNA ENZYME WITH MG2+-DEPENDENT RNA PHOSPHOESTERASE ACTIVITY
A DNA ENZYME WITH MG2+-DEPENDENT RNA PHOSPHOESTERASE ACTIVITY
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DOI：
10.1016/1074-5521(95)90028-4
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发表时间：
1995-10-01
影响因子：
--
通讯作者：
JOYCE, GF
中科院分区：
文献类型：
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作者：
BREAKER, RR;JOYCE, GF
背景：以前，我们证明了DNA可以作为一种酶，在Pb 2+依赖的RNA磷酸酯的切割。这是一个简单的反应，在pH 7.0和23 ℃下，在1 mM Pb（OAc）（2）存在下，典型RNA磷酸酯的非催化速率类似于10（-4）min（-1）。Mg 2+依赖的反应更困难，在可比条件下，未催化的速率类似于10（-7）min（-1）。Mg 2+依赖性切割与生物学具有特殊的相关性，因为它与细胞内条件相容。使用体外选择，我们试图开发一个在各种二价金属存在下操作的磷酸酯裂解DNA酶家族，特别关注Mg 2+依赖性反应。结果：我们产生了含有40个随机核苷酸的>10（13）个DNA的群体，并进行了多轮的选择性扩增，富集在1 mM Mg 2+、Mn 2+、Zn 2+或Pb 2+存在下切割靶RNA磷酸酯的分子。在第六轮之后对来自Mg 2+谱系的单个克隆的检查揭示了由三茎连接组成的催化基序。该基序被部分随机化，并进行另外七轮选择性扩增，以0.01 min（-1）的速率产生催化剂。优化的DNA催化剂被分为单独的底物和酶域，并显示出具有类似的活性水平下的多个营业额conditions.Conclusions：我们已经产生了一个Mg 2+依赖的DNA酶，切割目标RNA磷酸酯的催化速率类似于10（5）倍以上的未催化的反应。这种活性与细胞内条件相容，提高了DNA酶在体内工作的可能性。
Background: Previously we demonstrated that DNA can act as an enzyme in the Pb2+-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of similar to 10(-4) min(-1) in the presence of 1 mM Pb(OAc)(2) at pH 7.0 and 23 degrees C. The Mg2+-dependent reaction is more difficult, with an uncatalyzed rate of similar to 10(-7) min(-1) under comparable conditions. Mg2+-dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg2+-dependent reaction.Results: We generated a population of >10(13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg2+, Mn2+, Zn2+ or Pb2+. Examination of individual clones from the Mg2+ lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction. This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01 min(-1). The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions.Conclusions: We have generated a Mg2+-dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate similar to 10(5)-fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.