Puttaraju M, Been M D
Department of Biochemistry, Duke University Medical Center, Durham, NC-27710, USA.
SAAS Bull Biochem Biotechnol. 1996;9:77-82.
An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, and tested for self-splicing activity. Precursor RNAs from these constructs spliced efficiently and produced circular exons in vitro. Using group I permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA component of RNaseP from B. subtilis, the HIV TAR and a short HIV Rev-binding element were generated and tested for activity and stability. The activity of circular ribozymes is comparable to the linear counterparts with similar core sequences. Circular forms of the TAR and Rev-binding element showed specific binding to Tfr-38 and Rev(35-50) peptide, respectively. To explore the potential for using this methodology to express circular RNA in vivo, circular forms of the HDV ribozyme and RNaseP RNA were produced in E. coli. Analysis of total RNA indicated that the precursor RNA spliced efficiently and accurately to produce circular ribozymes. The activity of in vivo expressed circular ribozymes could be demonstrated indicating that they fold into active conformation. These results suggest that self-splicing group I PIE sequences could prove useful for expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in cells.
将鱼腥藻I组内含子在环5、环6和环8处进行环形排列，并测试其自我剪接活性。这些构建体的前体RNA在体外高效剪接并产生环状外显子。利用I组排列的内含子-外显子序列，生成了丁型肝炎病毒核酶、来自枯草芽孢杆菌的核糖核酸酶P的RNA组分、HIV TAR和短HIV Rev结合元件的环状形式，并测试了其活性和稳定性。环状核酶的活性与具有相似核心序列的线性对应物相当。TAR和Rev结合元件的环状形式分别显示出与Tfr-38和Rev(35-50)肽的特异性结合。为了探索使用这种方法在体内表达环状RNA的潜力，在大肠杆菌中产生了丁型肝炎病毒核酶和核糖核酸酶P RNA的环状形式。总RNA分析表明，前体RNA高效且准确地剪接以产生环状核酶。体内表达的环状核酶的活性可以得到证明，表明它们折叠成活性构象。这些结果表明，自我剪接的I组PIE序列可能被证明可用于在细胞中表达小的稳定环状核酶/诱饵竞争物或反义RNA。
