体外靶向HCMV UL97 mRNA片段的m1 - GS核酶的构建

Wen-Jun Zhang, Hong-Jian Li, Yue-Qin Li, Hua-Kun He, Dong-Sheng Tang, Xin Zhang, Tian-Hong Zhou
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引用次数: 0

摘要

通过将一个寡核苷酸(引导序列,GS)与大肠杆菌RNase P的催化亚基M1 RNA的3'端共价连接,构建了序列特异性M1GS核酶(M1- t3)。该工程核酶靶向HCMV蛋白激酶(UL97)的mRNA序列,并能在体外有效地切割mRNA片段。进一步研究了一些结构元件在M1基因组中的意义(如3' CCA尾部序列和M1 RNA 3'端与GS 5'端之间的桥接序列)。结果表明,突变M1- GS(即M1- t3 *)的88个核苷酸桥接序列显著提高了对底物的裂解活性。此外,3'CCA尾序列被证实是M1 GS核酶裂解活性的必要元件。我们在研究中获得的这些数据将有助于理解M1 GS RNA与其底物之间的相互作用,并将显著促进抗hcmv应用的通用基因靶向剂的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Construction of an effective m1 GS ribozyme targeting HCMV UL97 mRNA segment in vitro.

A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.

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