ARTIFICIAL RNA RESTRICTION ENZYMES

这个项目的目标是开发裂解rna的分子。 具有高度的序列特异性并显示出催化周转率 RNA(人工限制性内切酶)的裂解。人造的 限制酶将由金属的酯交换反应构成。 催化剂和序列互补的寡脱氧核苷酸 一种核糖核酸。这项提议的独特之处在于使用了金属 裂解核糖核酸的酯交换催化剂。核糖核酸的酯交换反应 催化的磷(V)取代反应的一个例子 金属离子；在这个反应中，一条核糖核酸被 打破了单一的磷-氧键。因为金属 酯交换催化剂专用于RNA，不切割DNA， 不产生可扩散的自由基来裂解RNA并显示催化作用 周转率，由这些催化剂构建的人造酶将具有 许多令人向往的特性。N-乙二胺-N，N‘-二乙酸酯和 亚氨基二乙酸酯将被连接到寡核苷酸上。系绳长度 和金属离子(铅(II)、镧(III)或锌(II)将按顺序变化 以优化特定的切割。关于寡核苷酸长度的实验 而切割反应的温度将使我们能够确定 可能发生催化周转的条件。特定于序列 将检测用人工限制性内切酶切割t-RNAphe， 而卵裂的位置将由标准的方案确定 核酸测序。此外，我们建议进一步开发一种新的 锌(II)、镁(II)或镍(II)络合物催化剂 大环多胺中含有一种侧链的吡啶。金属作为一种 路易斯酸可能与悬挂的吡啶协同催化作用 通过双功能机制的酯交换反应。以加速催化 RNA裂解，吡啶相对于金属的位置 通过改变吡啶的附着位置和 链接器臂的长度。从长远来看，这些人为的限制 酶将被开发用于基因的选择性抑制 表达和作为实验室操作RNA的试剂。

The goal of this project is the development of molecules that cleave RNA with a high degree of sequence-specificity and show catalytic turnover in the cleavage of RNA (artificial restriction enzymes). Artificial restriction enzymes will be constructed from metal transesterification catalyst and an oligodeoxynucleotide that is complementary in sequence to an RNA. The unique feature of this proposal is the use of metal transesterification catalysts to cleave RNA. Transesterification of RNA is an example of a substitution reaction at phosphorus(V) which is catalyzed by metal ions; in this reaction, a strand of RNA is neatly cleaved by breaking a single phosphorus-oxygen bond. Because of metal transesterification catalysts are specific for RNA and do not cleave DNA, do not produce diffusible radicals to cleave RNA and show catalytic turnover, artificial enzymes constructed from these catalysts will have many desirable properties. N-ethyl-ethylenediamine-N, N'-diacetate and iminodiacetate will be attached to oligodeoxynucleotides. Tether length and metal ion (lead(II), lanthanum(III) or zinc(II) will be varied in order to optimize specific cleavage. Experimentation with oligonucleotide length and the temperature of the cleavage reaction will enable us to determine conditions where catalytic turnover may occur. The sequence-specific cleavage of t-RNAphe with artificial restriction enzymes will be examined, and the site of cleavage will be determined by standard protocol for nucleic acid sequencing. In addition, we propose to further develop a new class of catalysts based on zinc(II), magnesium(II) or nickel(II) complexes of macrocyclic polyamines containing a pendant pyridine. The metal as a Lewis acid may work in concert with the pendant pyridine to catalyze transesterification by a bifunctional mechanism. To accelerate catalytic RNA cleavage, the position of the pyridine with respect to the metal will be changed by varying the position of attachment of the pyridine and the length of the linker arm. In the long term, these artificial restriction enzymes will be developed for use as selective inhibitors of gene expression and as reagents for laboratory manipulations of RNA.