Nucleotides-1 to-4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage
Nucleotides-1 to-4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage
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DOI：
10.1089/oli.1.2000.10.53
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发表时间：
2000-02-01
期刊：
影响因子：
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通讯作者：
Perreault, JP
中科院分区：
文献类型：
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作者：
Deschênes, P;Lafontaine, DA;Perreault, JP
在过去，由于底物特异性被认为仅由8个核苷酸决定，因此δ核酶作为治疗工具的使用受到限制。最近，我们已经积累的证据表明，切割位点上游的底物序列（其不参与与δ核酶的结合）似乎在选择合适的切割位点时是必不可少的。为了理解该区域在有效切割中的作用，我们合成了一系列在位置-1至-4处具有单个和多个突变的小底物，并使用模型反基因组δ核酶确定其切割的动力学参数。发现一些底物是未切割的，而其他底物在最低和最有效切割的底物之间显示出>60倍的相对特异性差异，从-1至-4的每个位置处的碱基对底物被切割的能力有不同的贡献。位置-1至-4的最佳序列被确定为-1HRHY-4（H = U、C或A）。这些结果揭示了新的功能，有助于三角洲核酶切割的底物要求，并应增加使用这种独特的核酶的兴趣。
In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides, Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding,vith the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme, Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates, The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY-4 (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.