Abstract EANA2025-61

Proteinoid is a polyamino acid synthesized from amino acids and their precursors in a thermally molten state. Proteinoid microspheres are spherical molecular assemblies formed on the micrometer scale by dissolving proteinoid in hot water and then cooling it to room temperature. These microspheres are considered functional molecular assemblies that represent a step toward primitive cells, based on their synthetic conditions and source materials. However, the emergence of early life requires the coexistence and function of proteins and nucleic acids. Last year, we demonstrated that nucleic acids can be incorporated into microspheres. The goal of this study was to confirm the expression of nucleic acids in microspheres. Specifically, we introduced a plasmid encoding green fluorescent protein (GFP) into the microspheres and then transformed them into Escherichia coli (E. coli) to examine GFP expression.

Proteinoid was synthesized by melting monoammonium malate in an anhydrous environment. A proteinoid microsphere suspension was prepared by dissolving proteinoid in hot water, then cooling it to room temperature after removing insoluble matter. This suspension was redissolved by heating, and the pGLO plasmid was added while cooling. Scanning electron microscopy (SEM) revealed that the pGLO plasmid-containing microspheres were perfectly spherical structures ranging in diameter from tens of nanometers to several micrometers. Additionally, elemental analysis mapping by electron probe microanalysis was used to observe the pGLO plasmid-containing microspheres. Signals of phosphorus derived from DNA incorporated into microspheres were detected at positions where they overlapped with the oxygen and nitrogen signals corresponding to the shape of the proteinoid microsphere. Therefore, the pGLO plasmid was encapsulated in proteinoid microspheres.

The pGLO plasmid-containing microspheres were added to an E. coli suspension without treating the cells to make them competent or subjecting them to heat shock. The suspension was then dropped onto an ampicillin- and arabinose-containing medium and cultured at 38°C. Only the E. coli containing microspheres could grow, while those without microspheres could not. Additionally, the E. coli containing the microspheres exhibited green fluorescence under a fluorescence microscope, while the untransformed E. coli did not.

In this study, we confirmed that nucleic acids can be expressed in E. coli when incorporated into microspheres. This finding is an important step toward understanding the coexistence and function of proteins and nucleic acids in the origin of life. This research deepens our understanding of the origin of life and has potential applications in biotechnology and genetic engineering, such as drug delivery.