Production of circular streptavidin RNA aptamer in vivo.
Production of circular streptavidin RNA aptamer in vivo.
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DOI：
10.1093/nass/nrm196
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发表时间：
2007-11
期刊：
影响因子：
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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治疗中最有希望的候选体之一。我们之前报道了体外生产环状链亲和素RNA适配体。在这里，我们展示了在体内产生环状RNA适体的应用。首先，我们构建了一个环状RNA表达载体，该载体包含T7启动子序列和T7终止子序列之间的自剪接排列内含子-外显子（PIE）序列，通过JM109（DE3）细胞中产生的T7 RNA聚合酶进行转录。通过添加IPTG触发T7 RNA聚合酶驱动的RNA表达，并从生成的PIE转录本中生成环状RNA。JM109（DE3）细胞生成的环状链亲和素RNA适配体，采用溴化乙啶染色进行二维变性PAGE分析。利用自连接序列特异性寡核苷酸探针进行的Northern blot分析和测序分析表明，大肠杆菌中精确地发生了自剪接和环状化过程。用固相DNA探针法从部分纯化的总RNA片段中很容易纯化环状适体。这是环状RNA适体在体内的首次表达和纯化，为低成本生产RNA适体铺平了新的道路。
RNA aptamers is one of highly hopeful candidates for RNA therapeutics. We previously reported an in vitro production of a circular streptavidin RNA aptamer. Here we show an application for producing the circular RNA aptamer in vivo. First, we constructed a circular RNA expression vector that contained self-splicing permuted intron-exon (PIE) sequences between T7 promoter and T7 terminator sequences so as to be transcribed by T7 RNA polymerase produced in JM109(DE3) cells. RNA expression driven by T7 RNA polymerase was triggered by addition of IPTG and the circularized RNA was generated from the resulting PIE transcripts. Circular streptavidin RNA aptamer generated in the JM109(DE3) cells was detected by a two dimensional denaturing PAGE analysis using the ethidium bromide staining. Northern blot analysis using a self-ligated sequence specific oligo DNA probe and sequencing analysis revealed that the self-splicing and circularization process precisely occurred in E. coli. The circular aptamer was easily purified by a solid phase DNA probe method from a partially purified total RNA fraction. This is the first demonstration of an in vivo expression of a circular RNA aptamer and its purification, paving the new way for inexpensive production of RNA aptamer.