Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA.
Biomacromolecules. 2012 Oct 8;13(10):3395-400. doi: 10.1021/bm301155w. Epub 2012 Sep 28.
Lipid-coated DNA nanoparticles (lipoplexes) are a powerful gene delivery tool with promising therapeutic applications. The mechanism of lipoplex assembly remains poorly understood. We explored DNA packing by a cationic lipid DSTAP (distearoyl trimethylammonium-propane) using magnetic tweezers. DSTAP-induced DNA condensation occurred as a series of bursts with the mean step size of 60-80 nm. The pause time preceding the steps could be approximated as a bimodal distribution, which reveals at least two distinct condensation pathways. The rapidly condensed DNA was more resilient to force-induced decondensation. The proportion of the stable, fast-formed complexes decreased at high salt concentrations. A similar trend was observed in bulk experiments. Lipoplexes assembled at low salt concentration more efficiently shielded DNA from fluorescent dyes and DNase even after transfer to the high salt conditions. These data reveal that lipoplex folding occurs via two parallel pathways even at the single molecule level. The progress through the two pathways can be monitored in real time using single DNA manipulations. The relative efficiency of the two pathways can be varied by external conditions.
脂质体 DNA 纳米颗粒（脂质体）是一种强大的基因传递工具，具有有前途的治疗应用。脂质体组装的机制仍知之甚少。我们使用磁镊探索了阳离子脂质 DSTAP（二硬脂酰基三甲基铵丙烷）对 DNA 的包装。DSTAP 诱导的 DNA 缩合发生一系列爆发，平均步长为 60-80nm。在步骤之前的暂停时间可以近似为双峰分布，这表明至少存在两种不同的缩合途径。快速凝结的 DNA更能抵抗力诱导的去凝结。在高盐浓度下，稳定的快速形成复合物的比例降低。在体实验中也观察到了类似的趋势。在低盐浓度下组装的脂质体更有效地保护 DNA 免受荧光染料和 DNase 的影响，即使在转移到高盐条件下也是如此。这些数据表明，即使在单分子水平上，脂质体的折叠也通过两条平行途径发生。通过单 DNA 操作可以实时监测两条途径的进展。两条途径的相对效率可以通过外部条件来改变。
