Encapsulation of Nucleic Acids into Giant Unilamellar Vesicles by Freeze-Thaw: a Way Protocells May Form

Abstract

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.

Macrobiont: Cradle for the Origin of Life and Creation of a Biosphere

“…The formation reactions of nucleotides from their constituent nucleobases, ribose, and phosphate produce H 2 O and hence are favored under conditions that reduce water activity. Most importantly, the facilitation of the polymerization reactions necessary to proceed from nucleotides to RNA or DNA and from amino acids to proteins require dehydration reactions, which have been demonstrated to be enabled by wet-dry cycles [42][43][44][45] or also by freeze-thaw cycling [46][47][48][49][50][51]. In appropriate climates, such a pond may be subject to both wet-dry and freeze-thaw phenomena, thus strongly promoting the dehydration reactions for macromolecule polymerization by each of these distinct phenomena.…”

“…Ice formation causes excretion of solubilized constituents, which increases their concentration in the residual liquid. This provides ample opportunity for freeze-thaw phenomena, an area of increased interest as a medium for quasi-compartmentalization [51,52] as well as formation of nucleotide precursors, and their condensation into RNA oligomers [47,52].…”

“…As advocated in various studies, the earliest form of compartmentalization could have been leaky coacervates [108], mineral surfaces [109], pores in hydrothermal vent chimneys [113,114], sulfide bubbles [115], vesicles from amphiphiles [6,15,51], and/or eventually lipid membranes [6,45,94]. Different versions may have played a role in rudimentary life before today's versions arose.…”

“…Life 2020, 10, x FOR PEER REVIEW 38 of 58 sulfide bubbles [115], vesicles from amphiphiles [6,15,51], and/or eventually lipid membranes [6,45,94]. Different versions may have played a role in rudimentary life before today's versions arose.…”

Primitive Compartmentalization for the Sustainable Replication of Genetic Molecules

“…In addition, freezing or F-T cycles supported other important processes for the emergence of life, such as synthesis of RNA through non-enzymatic polymerization of activated nucleotides [78][79][80], formation of longer RNAs through non-enzymatic ligation and recombination of short fragments [81,82], and assembly and catalysis of ribozymes [39,[83][84][85]. F-T cycles have also been demonstrated to induce the encapsulation of genetic molecules in phospholipid vesicles [48,49], their inter-vesicle exchange [86], and sustainable RNA replication through fusion-division of vesicles [87]. Thus, ice environments seem to be plausible sites for the development of early life.…”