The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA
The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA
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DOI：
10.1016/s0167-4781(97)00069-9
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发表时间：
1997-08-07
期刊：
影响因子：
--
通讯作者：
Baker, JR
中科院分区：
文献类型：
--
作者：
Bielinska, AU;KukowskaLatallo, JF;Baker, JR
与基于病毒的系统相比，应用合成载体进行基因转移具有潜在的优势。然而，人们对合成材料与DNA结合的机制以及这种相互作用产生的DNA复合物的性质知之甚少。聚酰胺胺（PAMAM）树状大分子是一种具有明确球形结构的独特聚合物。树状大分子结合DNA形成复合物，在体外有效地转染细胞。我们检查了DNA/树突复合物的形成，发现它完全基于电荷相互作用。复合物的电子显微镜检查表明，大部分质粒DNA被收缩成孤立的环状体，但也显示出更大的，不规则的聚合物和DNA聚集。质粒DNA与树状大分子的结合似乎改变了二级和三级结构，但不会使DNA断裂或改变其一级结构。复杂的DNA可以通过特定的核酸酶或含有核酸酶活性的细胞提取物来防止降解。虽然树突状复合物DNA启动子（T7聚合酶或真核RNA聚合酶II）在体外的转录起始受到抑制，但RNA转录的延伸和翻译似乎不受影响。当与非乙酰化组蛋白等自然发生的多阳离子络合时，这些类似于DNA功能的改变，然而，与树状大分子络合的DNA似乎保持转录活性，而类似电荷比的组蛋白复合物则没有。这些结果阐明了PAMAM树突状聚合物与DNA相互作用的某些方面。并可能导致改进聚合物的设计或DNA复合物的形成，从而提高非病毒基因转移的效率。(C) 1997爱思唯尔科学有限公司
The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. However, little is known about the mechanisms involved in binding of synthetic materials to DNA and the nature of the DNA complexes that result from this interaction. Polyamidoamine (PAMAM) dendrimers are unique polymers with defined spherical structure. Dendrimers bind DNA to form complexes that efficiently transfect cells in vitro. We examined the formation of DNA/dendrimer complexes and found it based entirely on charge interaction. Electronmicroscopic examination of the complexes indicated that the majority of the plasmid DNA is contracted into isolated toroids, but also revealed larger, irregular aggregates of polymer and DNA. The binding of plasmid DNA to dendrimer appears to alter the secondary and tertiary structure, but does not fragment the DNA or alter its primary structure. Complexed DNA is protected against degradation by either specific nucleases or cellular extracts containing nuclease activity. While the initiation of transcription in vitro from promoters (for either T7 polymerase or eukaryotic RNA polymerase II) in dendrimer-complexed DNA is inhibited, elongation of the RNA transcript and translation do not appear to be affected. These resemble alterations of the DNA function when complexed with naturally-occurring polycations like non-acetylated histones, However, DNA complexed to dendrimer appears to maintain transcriptional activity while histone complexes at similar charge ratios do not, These results elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation of DNA complexes that will increase the efficiency of non-viral gene transfer. (C) 1997 Elsevier Science B.V.