Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection
Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection
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DOI：
10.1016/j.jconrel.2005.08.019
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发表时间：
2005-11-28
影响因子：
10.8
通讯作者：
Sakurai, K
中科院分区：
文献类型：
--
作者：
Anada, T;Karinaga, R;Sakurai, K
我们以前的工作表明，天然β-（1 -> 3）-D-葡聚糖-β-葡聚糖（SPG）可以与单链寡核苷酸（ssODNs）形成稳定的复合物。将蛋白转导肽连接到SPG上，并与ssODNs复合，得到的复合物可诱导结合ODNs的细胞转染，而不产生严重的细胞毒性。然而，没有可用的技术来使用SPG检测双链DNA（dsDNA）或质粒DNA。本文介绍了一种新的方法来转染双链DNA，显示制备和转染效率的最小尺寸的基因具有环状聚（dA）（80）两端。这种dsDNA的poly（dA）环可以与SPG形成复合物。将具有poly（dA）环的siRNA编码dsDNA与Tat连接的SPG复合以沉默荧光素酶表达。当LTR启动子控制下可表达荧光素酶的LTR-Luc-HeLa细胞暴露于该复合物时，荧光素酶的表达被抑制（即，RNAi效应增强）。细胞毒性研究表明，与聚乙烯亚胺相比，Tat-SPG复合物诱导的细胞死亡少得多，表明所提出的方法比常规方法造成的伤害小。Tat-SPG/poly（dA）环状dsDNA复合物具有与病毒基因组相似的结构，因为dsDNA末端能够诱导转染和保护。目前的工作确定了SPG和聚（dA）环的最小尺寸的基因组合作为一个无毒的基因传递系统的候选人。(c)2005 Elsevier B. V.保留所有权利。
Our previous work showed that a natural beta-(1 -> 3)-D-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)(80) on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system. (c) 2005 Elsevier B.V. All rights reserved.