In vitro and in vivo production and purification of circular RNA aptamer.
In vitro and in vivo production and purification of circular RNA aptamer.
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DOI：
10.1016/j.jbiotec.2008.12.012
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发表时间：
2009-02
影响因子：
4.1
通讯作者：
S. Umekage;Y. Kikuchi
中科院分区：
文献类型：
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作者：
S. Umekage;Y. Kikuchi
RNA适体是RNA治疗剂的潜在候选物。它们必须在临床上进行修饰以用于医学应用，因为它们易受固有核糖核酸酶的影响。由于没有任何化学修饰的环状RNA分子在细胞提取物中比线性RNA分子稳定得多，我们报告了在体外和体内产生环状形式的链霉亲和素RNA适体。环化是通过来自T4噬菌体基因td的自拼接排列的内含子-外显子序列完成的。该序列在大肠杆菌细胞和体外均可产生。与线性形式的链霉亲和素适体相比，环化的链霉亲和素RNA适体保留了其与链霉亲和素的结合，并且在HeLa细胞提取物中是稳定的。从E.在排除凝胶电泳的阴离子交换层析之后，使用固相DNA探针法从总RNA级分纯化大肠杆菌细胞。本研究为设计和纯化有用的RNA适体提供了另一种方法。
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.