In vitro selection of aptamers that act with Zn2+
In vitro selection of aptamers that act with Zn2+
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DOI：
10.1016/s0162-0134(00)00158-6
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发表时间：
2000-11-01
影响因子：
3.9
通讯作者：
Sugimoto, N
中科院分区：
文献类型：
--
作者：
Kawakami, J;Imanaka, H;Sugimoto, N
用Zn 2+进行体外选择以分离与HIV-1达特蛋白结合的新型RNA分子，锌依赖性适体。通过使用硝酸纤维素过滤器从随机RNA文库中收集与达特结合的RNA，并通过随后的逆转录、聚合酶链反应和转录再生为下一代RNA文库。在6轮和12轮选择后确定所选RNA的序列。用Mg 2+进行正常选择程序后的对照克隆具有类似于TAR或Arg适体的必需序列的共有UUG。另一方面，从用Zn 2+筛选的文库中发现了许多独特的序列，并且具有最丰富序列的RNA（克隆31）仅在Zn 2+存在时才与达特紧密结合。克隆31 RNA的二级结构预测通过使用我们的热力学参数和RNA的酶切的计算预测。制备了几种模型RNA，并研究了这些RNA与达特的结合特性。结果，所有的模型RNA都没有再现克隆31的结合特性。因此，与Zn 2+作用的达特适体应该需要相对较长的序列区域，其能够提供用于结合的几个基序的三级合作。(C)2000 Elsevier Science B. V.保留所有权利。
An in vitro selection was carried out with Zn2+ to isolate novel RNA molecules, zinc-dependent aptamers, that bind to HIV-1 Tat protein. RNAs bound to Tat were collected by using a nitrocellulose filter from a library of random RNAs and regenerated to the next generation of the RNA library by subsequent reverse transcription, polymerase chain reaction, and transcription. Sequences of the selected RNAs were determined after 6 and 12 rounds of the selection. The control clones after normal selection procedure with Mg2+ had a consensus UUG that resembled essential sequences of TAR or Arg aptamers. On the other hand, many unique sequences were revealed from a library selected with Zn2+ and the RNA with most abundant sequence (clone 31) bound to Tat tightly only when Zn2+ existed. The secondary structure of clone 31 RNA was predicted by using a computational prediction with our thermodynamic parameters and enzymatic scission of the RNA. Several model RNAs were prepared and the binding property of these RNAs to Tat were investigated. As a result, all the model RNAs did not reproduce the binding property of clone 31. Therefore, the Tat aptamer that acts with Zn2+ should require a relatively longer region of the sequence which, is able to offer tertiary cooperation of several motifs for the binding. (C) 2000 Elsevier Science B.V. All rights reserved.