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通过滚环扩增和酶切高效扩增自凝胶化多足状结构DNA

Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion.

作者信息

Yata Tomoya, Takahashi Yuki, Tan Mengmeng, Hidaka Kumi, Sugiyama Hiroshi, Endo Masayuki, Takakura Yoshinobu, Nishikawa Makiya

机构信息

Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan.

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan,

出版信息

Sci Rep. 2015 Oct 14;5:14979. doi: 10.1038/srep14979.

Abstract

The application of DNA as a functional material such as DNA hydrogel has attracted much attention. Despite an increasing interest, the high cost of DNA synthesis is a limiting factor for its utilization. To reduce the cost, we report here a highly efficient amplification technique for polypod-like structured DNA (polypodna) with adhesive ends that spontaneously forms DNA hydrogel. Two types of polypodna with three (tripodna) and four (tetrapodna) pods were selected, and a template oligodeoxynucleotide, containing a tandem sequence of a looped tripodna or tetrapodna, respectively, along with restriction enzyme (TspRI) sites, was designed. The template was circularized using T4 DNA ligase, and amplified by rolling circle amplification (RCA). The RCA product was highly viscous and resistant to restriction digestion. Observation under an electron microscope revealed microflower-like structures. These structures were composed of long DNA and magnesium pyrophosphate, and their treatment with EDTA followed by restriction digestion with TspRI resulted in numerous copies of polypodna with adhesive ends, which formed a DNA hydrogel. Thus, we believe this technique provides a new approach to produce DNA nanostructures, and helps in expanding their practical applications.

摘要

DNA作为一种功能材料,如DNA水凝胶的应用已引起广泛关注。尽管人们对其兴趣日益浓厚,但DNA合成的高成本是其应用的限制因素。为了降低成本,我们在此报告一种高效的扩增技术,用于具有粘性末端的多足状结构DNA(多足DNA),该DNA可自发形成DNA水凝胶。我们选择了具有三个(三脚架DNA)和四个(四脚架DNA)足的两种类型的多足DNA,并设计了一种模板寡脱氧核苷酸,其分别包含环状三脚架DNA或四脚架DNA的串联序列以及限制性内切酶(TspRI)位点。使用T4 DNA连接酶将模板环化,并通过滚环扩增(RCA)进行扩增。RCA产物具有高粘性且抗限制性消化。电子显微镜观察显示出微花状结构。这些结构由长DNA和焦磷酸镁组成,用EDTA处理后再用TspRI进行限制性消化,得到了大量具有粘性末端的多足DNA拷贝,这些拷贝形成了DNA水凝胶。因此,我们相信该技术为生产DNA纳米结构提供了一种新方法,并有助于扩大其实际应用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b580/4604513/a4bf4eca05a2/srep14979-f1.jpg

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