Bhattacharya S, Mandal S S
Department of Organic Chemistry, Indian Institute of Science, Bangalore.
Biochemistry. 1998 May 26;37(21):7764-77. doi: 10.1021/bi971772j.
Complex formation of DNA with a number of cationic amphiphiles has been examined using fluorescence, gel electrophoresis, and chemical nuclease digestion. Here we have addressed the status of both DNA and lipid upon complexation with each other. DNA upon binding with cationic amphiphiles changes its structure in such a way that it loses the ability to intercalate and becomes resistant to nuclease digestion. Fluorescence anisotropy measurements due to 1, 6-diphenylhexatriene (DPH) doped in cationic liposomes demonstrated that upon complexation with DNA, the resulting complexes still retain lamellar organizations with modest enhancement in thermal stabilities. The lipid-DNA complexation is most effective only when the complexation was carried out at or around the phase transition temperatures of the cationic lipid employed in the complexation with DNA. The release of DNA from cationic lipid-DNA complexes could be induced by several anionic additives. Determination of fluorescence anisotropies (due to DPH) as a function of temperature clearly demonstrates that the addition of equivalent amounts of anionic amphiphile into cationic lipid-DNA complexes leads to the ion-pairing of the amphiphiles, the melting profiles of which are virtually the same as those obtained in the absence of DNA. In this process DNA gets released from its complexes with cationic lipids and regains its natural intercalation ability, movement, and staining ability on agarose gel and also the sensitivities toward nuclease digestion. This clearly suggests that combination of ion-pairing and hydrophobic interactions between cationic and anionic amphiphiles is stronger than the electrostatic forces involved in the cationic lipid-DNA complexation. It is further revealed that the DNA release by anions is most efficient from the cationic lipid-DNA complexes at or around the Tm of the cationic lipid used in DNA complexation. This explains why more effective DNA delivery is achieved with cationic lipids that bear unsaturated hydrocarbon chains than with their saturated hydrocarbon counterparts.
已使用荧光、凝胶电泳和化学核酸酶消化法研究了DNA与多种阳离子两亲物的复合物形成情况。在此，我们探讨了DNA和脂质相互复合时各自的状态。DNA与阳离子两亲物结合后，其结构发生变化，失去了嵌入能力，并变得对核酸酶消化具有抗性。对掺杂在阳离子脂质体中的1,6 - 二苯基己三烯（DPH）进行荧光各向异性测量表明，与DNA复合后，所得复合物仍保留层状结构，热稳定性略有提高。脂质 - DNA复合作用只有在与DNA复合所用阳离子脂质的相变温度或其附近进行时才最有效。几种阴离子添加剂可诱导DNA从阳离子脂质 - DNA复合物中释放。测定荧光各向异性（归因于DPH）随温度的变化清楚地表明，向阳离子脂质 - DNA复合物中加入等量的阴离子两亲物会导致两亲物的离子配对，其熔解曲线与在无DNA情况下获得的熔解曲线几乎相同。在此过程中，DNA从其与阳离子脂质的复合物中释放出来，恢复其天然的嵌入能力、迁移能力以及在琼脂糖凝胶上的染色能力，同时也恢复了对核酸酶消化的敏感性。这清楚地表明，阳离子和阴离子两亲物之间的离子配对和疏水相互作用的组合比阳离子脂质 - DNA复合作用中涉及的静电力更强。进一步揭示，在DNA复合所用阳离子脂质的Tm或其附近，阴离子从阳离子脂质 - DNA复合物中释放DNA的效率最高。这解释了为什么带有不饱和烃链的阳离子脂质比其饱和烃对应物能实现更有效的DNA递送。
