Bridge Sequence对M1GS核酶体外切割活性的影响

目的探讨B ridge Sequence(桥序列)对M1GS核酶体外切割活性的影响,从而为人工M1GS核酶的优化设计提供一定的理论依据。方法以含大肠杆菌核酶P催化单位(M1 RNA)基因的pFL117质粒作为模板,通过巧妙设计不同的下游引物,经PCR扩增、扩增产物克隆及克隆基因的体外转录,构建一组具有不同桥序列的人工M1GS核酶。进一步将所构建的上述人工M1GS核酶分别与同位素标记的底物RNA片段进行体外切割试验,并通过同位素扫描成像系统对各M1GS核酶的切割产物加以分析。结果成功构建了针对HCMV UL54mRNA T7靶位的、四种不同桥序列长度的M1GS核酶,其桥序列长度分别为0n、t6nt、20nt和88nt,并依次命名为M1GS-T7(0)、M1GS-T7(6)、M1GS-T7(20)和M1GS-T7(88)。经体外切割试验证实,M1GS-T7(88)的靶向切割活性最强,M1GS-T7(20)的切割活性相对较弱,而M1GS-T7(0)及M1GS-T7(6)则无明显的切割活性。结论人工M1GS核酶中桥序列的长度对于其体外切割活性具有重要影响,提示在人工M1GS核酶的优化设计时,桥序列的长度是不可忽视的因素之一。

The influence of bridge sequence on the cleavage activity of M1GS ribozyme in vitro

Objective To elucidate the influence of the length of bridge sequence in M1GS ribozyme on its cleavage activity in vitro.Methods Using the pFL117 plasmid,which containing M1 gene of the RNase P from Escherichia coli as the template,DNA products encoding the artificial M1GS gene which contain different lengths of bridge sequences were amplified with PCR method.By further cloning and transcribing in vitro with these PCR products,a group of artificial M1GS ribozymes with different lengths of bridge sequences were constructed.Furthermore,the cleavage activities of these artificial M1GS ribozymes aimed to the ~32P-labelled substrate were examinated with in vitro cleavage reaction and were analyzed with the Typhoon 9200.Results The four M1GS ribozymes targeting to the UL54 gene of HCMV,which containing a different length of bridge sequence,were successfully constructed and named M1GS-T7(0),M1GS-T7(6),M1GS-T7(20),M1GS-T7(88),accordingly.The cleavage activity of M1GS-T7(88) was highest,and the activity of M1GS-T7(20) was relatively lower.While both of the activities of M1GS-T7(0) and M1GS-T7(6) were not obviously observed.These results showed that four M1GS ribozymes with different lengths of bridge sequences(0nt,6nt,20nt and 88nt) had different cleavage activities on the substrate in vitro.Conclusions The length of bridge sequence was an important factor to the in vitro cleavage activity of the artificial M1GS ribozyme.This study will be helpful in understanding the interaction between the M1GS RNA and its substrates,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.