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In vitro and in vivo production and purification of circular RNA aptamer

机译:环状RNA适体的体外和体内生产与纯化

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摘要

RNA aptamers are potential candidates for RNA therapeutics. They must be clinically modified for medical applications because they are vulnerable to indigenous ribonucleases. Since circular RNA molecules without any chemical modification are much more stable than linear ones in a cell extract, we report the production of a circular form of streptavidin RNA aptamer both in vitro and in vivo. Circularization was accomplished by self-splicing permuted intron-exon sequences derived from T4 bacteriophage gene td. This sequence was producible in both Escherichia coli cells and in vitro. The circularized streptavidin RNA aptamer retained its binding of streptavidin and was stabile in HeLa cell extracts compared to the linear form of the streptavidin aptamer. The self-spliced circular RNA from the transcribed permuted intron-exon transcripts in E. coli cells was purified from a total RNA fraction using the solid-phase DNA probe method following anion exchange chromatography that excluded gel electrophoresis. This study provides an alternative method for designing and purifying useful RNA aptamers.
机译:RNA适体是RNA治疗剂的潜在候选者。由于它们易受本地核糖核酸酶的影响,因此必须对其进行临床修饰以用于医学应用。由于在细胞提取物中未经任何化学修饰的环状RNA分子比线性分子稳定得多,因此我们报道了在体外和体内均产生环状链霉亲和素RNA适体。通过自剪接来自T4噬菌体基因td的排列的内含子外显子序列来完成环化。该序列在大肠杆菌细胞和体外均可产生。与链霉抗生物素蛋白适体的线性形式相比,环化的链霉抗生物素蛋白RNA适体保留了其与链霉抗生物素蛋白的结合并且在HeLa细胞提取物中稳定。在排除凝胶电泳的基础上,采用阴离子交换色谱法,使用固相DNA探针法从大肠杆菌总RNA中纯化出自转录的内含子-外显子转录物的自剪接环状RNA。这项研究为设计和纯化有用的RNA适体提供了另一种方法。

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