滚环转录结合定点切割制备小RNA的技术探究  

Study on the method of small RNAs preparation using rolling circle transcription and site-specific disconnection

作　　者：李灿[1] 王星宇[1] 王静[1] 潘孝明[1] 董平[1] 梁兴国[1] 

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266003

出　　处：《生物技术》2016年第3期245-251,共7页Biotechnology

基　　金：国家自然科学基金项目(No.31571937;No.31201327);山东省博士后基金

摘　　要：[目的]优化滚环转录及定点酶切中关键技术,提高小RNA合成量。[方法]通过在引物与模板之间设计不互补部分,形成泡状凸起,引发滚环转录;使用天然DNAzyme 10-23定点剪切转录产物;另外改变RNase H酶切中Aid-DNA修饰核酸数目及位置,指导RNase H切割;DNaseⅠ降解模板DNA后,不经高温失活,直接进入RNase H酶切体系。[结果]由凸起结构引发转录,转录效率提高约5倍;RNase H在仅有3个修饰核酸间隔分布的Aid-DNA-3b辅助下,可高效定点剪切,而DNAzyme 10-23无法充分切割滚环转录产物;未失活的DNaseⅠ对Aid-DNA-3b的降解仅占8.7%,可直接进入RNase H酶切体系。[结论]引入凸起结构可提高转录效率约5倍;DNaseⅠ可直接进入酶切体系,随后使用Aid-DNA-3b介导酶切,产量可提高1.4倍。[ Objective ] To improve the yield of small RNA synthesis by optimizing key procedures during rolling circle tran- scription and site - specific disconnection. [ Methods ] The transcription was initiated from transcription bubble which was formed by partly unpaired template and primer. Then the large size RNA was specific cleaved by DNAzyme 10 - 23. And em- ployed the Aid - DNAs containing modified nucleotides with different number and position to guide RNase H site - specific dis- connection. The template DNA was digested by DNase I and the product was directly cleaved by RNase H without high temper- ature inactivation of DNase I [ Results] The transcription efficiency was improved by ~ 5 folds due to the formation of bubble. RNase H could effectively cleave RNA product when three interval distributed modified nucleotides was existed in Aid - DNA, however,the DNAzyme I0 -23 could not effectively cleave the transcription product. The inactivation of DNase I caused 8.7% Aid -DNA -3b loss thus could be omitted. [ Conclusion] The transcription efficiency was improved -5 fold by introdu- cing the bubble. The inactivation of DNase I with high temperature could be omitted and the final yield could be improved by 1.4 fold with the help of Aid - DNA - 3b.

关 键 词：滚环转录 定点切割 DNAZYME RNASE H DNaseⅠ 小RNA 

分 类 号：Q789[生物学—分子生物学]