Qiao Hai, Hu Na, Bai Jin, Ren Lili, Liu Qing, Fang Liaoqiong, Wang Zhibiao
State Key Laboratory of Ultrasound Engineering in Medicine Co-Founed by Chongqing and the Ministry of Science and Technology, Chongqing Key Laboratory of Biomedical Engineering, College of Biomedical Engineering, Chongqing Medical University, P. O. Box 153, No.1 Yixueyuan Road, Yuzhong District, Chongqing, 400016, People's Republic of China.
Orig Life Evol Biosph. 2017 Dec;47(4):499-510. doi: 10.1007/s11084-016-9527-9. Epub 2016 Nov 2.
Protocells are believed to consist of a lipid membrane and encapsulated nucleic acid. As the lipid membrane is impermeable to macromolecules like nucleic acids, the processes by which nucleic acids become encapsulated inside lipid membrane compartments are still unknown. In this paper, a freeze-thaw method was modified and applied to giant unilamellar vesicles (GUVs) and deoxyribonucleic acid (DNA) in mixed solution resulting in the efficient encapsulation of 6.4 kb plasmid DNA and similar length linear DNA into GUVs. The mechanism of encapsulation was followed by observing the effect of freeze-thaw temperatures on GUV morphological change, DNA encapsulation and ice crystal formation, and analyzing their correlation. Following ice crystal formation, the shape of spherical GUVs was altered and membrane integrity was damaged and this was found to be a necessary condition for encapsulation. Heating alone had no effects on DNA encapsulation, but was helpful for restoring the spherical shape and membrane integrity of GUVs damaged during freezing. These results suggested that freeze-thaw could promote the encapsulation of DNA into GUVs by a mechanism: the vesicle membrane was breached by ice crystal formation during freezing, DNA entered into damaged GUVs through these membrane gaps and was encapsulated after the membrane was resealed during the thawing process. The process described herein therefore describes a simple way for the encapsulation of nucleic acids and potentially other macromolecules into lipid vesicles, a process by which early protocells might have formed.
原细胞被认为由脂质膜和包裹其中的核酸组成。由于脂质膜对核酸等大分子是不可渗透的，核酸如何被包裹在脂质膜隔室内的过程仍然未知。在本文中，一种冻融方法被改进并应用于混合溶液中的巨型单层囊泡（GUV）和脱氧核糖核酸（DNA），从而实现了将6.4 kb的质粒DNA和类似长度的线性DNA高效包裹进GUV。通过观察冻融温度对GUV形态变化、DNA包裹和冰晶形成的影响，并分析它们之间的相关性，来探究包裹机制。冰晶形成后，球形GUV的形状发生改变，膜完整性受损，这被发现是包裹的必要条件。单独加热对DNA包裹没有影响，但有助于恢复在冷冻过程中受损的GUV的球形形状和膜完整性。这些结果表明，冻融可以通过一种机制促进DNA包裹进GUV：在冷冻过程中，冰晶形成使囊泡膜破裂，DNA通过这些膜间隙进入受损的GUV，并在解冻过程中膜重新密封后被包裹。因此，本文所述过程描述了一种将核酸以及潜在的其他大分子包裹进脂质囊泡的简单方法，这可能是早期原细胞形成的过程。
