ARTIFICIAL RNA RESTRICTION ENZYMES

该项目的目标是开发切割RNA的分子 具有高度的序列特异性，并在 RNA的切割（人工限制酶）。 人工 限制酶将由金属酯交换反应构建 催化剂和寡脱氧核苷酸，所述寡脱氧核苷酸在序列上与 RNA。 这一提案的独特之处在于使用了金属 酯交换催化剂切割RNA。 RNA的酯交换作用是 在磷（V）上的取代反应的一个实例， 在这个反应中，一条RNA链被金属离子整齐地切割， 断开一个磷氧键 因为金属 酯交换催化剂对RNA是特异性的并且不裂解DNA， 不产生可扩散的自由基来切割RNA， 营业额，由这些催化剂构建的人工酶将具有 许多令人满意的特性。N-乙基-乙二胺-N，N '-二乙酸盐和 亚氨基二乙酸将连接到寡脱氧核苷酸上。 系绳长度 和金属离子（铅（II）、镧（III）或锌（II））将按顺序变化 以优化特异性切割。 寡核苷酸长度实验 裂解反应的温度可以让我们确定 可能发生催化转化的条件。 序列特异 将检测用人工限制酶切割t-RNAphe， 切割位点将通过标准方案确定， 核酸测序。 此外，我们建议进一步发展新的 一类基于锌（II）、镁（II）或镍（II）络合物的催化剂 含有吡啶侧基的大环多胺。 金属作为 刘易斯酸可与侧基吡啶协同作用以催化 通过双功能机制进行酯交换。 加速催化剂 RNA裂解时，吡啶相对于金属的位置将 可以通过改变吡啶和吡啶的连接位置来改变。 从长远来看，这些人为的限制 酶将被开发用作基因的选择性抑制剂， 表达和作为试剂用于RNA的实验室操作。

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.