DNA Crown Cells: Synthesis and Self-Replication

Shoshi Inooka* The Institute of Japan Applied Food Materials Science, Japan Association of Special Scientists, Japan 3-8-2 Nakada, Taihaku-ku Sendai 981-1104, Japan Corresponding Author: Shoshi Inooka, The Institute of Japan Applied Food Materials Science, Japan Association of Special Scientists, Japan 3-8-2 Nakada, Taihaku-ku Sendai 981-1104, Japan. Tel: +81-22-241-0795, Fax: +81-22-241-1146, E-mail: s3inooka@aol.com Citation: Shoshi Inooka (2017), DNA Crown Cells: Synthesis and Self-Replication. Int J Biotech & Bioeng. 3:1, 30-32. DOI:10.25141/2475-3432-2017-1.0030 Copyright: ©2017 Shoshi Inooka. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited


Introduction
Artificial cells, including liposomes, nanoparticles, and microcapsules, have been studied since the 1960s and have led to impressive achievements in the generation of artificial cells (Zhang et al, 2008;Lin et al. 2013; Kuruma et al. 2009). However, to date, artificial cells which can replicate autonomously have not been reported.
Recent work on artificial cells has focused on cell division or replication (Noireaux et al, 2011). I have developed a method to prepare artificial cells (Inooka, 2012;Inooka, 2016, Ref.5). The artificial cells are prepared by incubating in egg-white artificial cell seeds comprising sphigosine (Sph), DNA, and components that bind Sph-DNA, such as adenosine or uridine Ref.7). I previously reported (Inooka, 2016, Ref;8) that Sph-DNA aggregated with adenosine-lipids contained in egg-white to generate DNA crown cells. Here, I attempted to prepare DNA crown cells using a known and commercially available lipid, monolaurin. First, adenosine-lipids were prepared, then DNA crown cells were constructed using the resulting aggregates of Sph-DNA. The procedures for synthesizing DNA crown cells are described below.

Materials and Methods
Sph (Sigma, USA), DNA (Escherichia coli strain B, Sigma, USA), Adenosine (Sigma, USA and Wako, Japan), monolaurin (Tokyo Kasei, Japan), and white Leghorn eggs purchased from a market. Preparation of the compound from by mixing adenosine and monolaurin (A-M) A case to prepare A-M is as follows: 0.4 ml (0.1 M) monolarurin was added to 0.4 ml (0.1M) of adenosine solution. After mixing, 0.15 ml of ethanol was added to the mixture and the precipitate was collected and dried. The A-M precipitate was resolved in 1.0 ml distilled water and used. Effect of compound (A-M) on Sphingosine-DNA To examine the effect of A-M on Sph-DNA, Sph (90μl, 10mM) was added to 40μl/μl of DNA (1.7μg/μl). After heating the mixture, A-M solution (50μl) was added., then one drop of ethidium bromide solution was added to one drop of the Sph-DNA-A-M mixture. Then , a drop was placed on a glass slide and observed using phase contrast microscopy and fluorescence microscopy.

Abstract
It has been half a century since the first studies on cell biology. During this time, it was shown that cells consist of a membrane made of lipid-polymer complexes comprising proteins and carbohydrates complexed with lipids. Further studies on cell biology have been based according to this understanding of the structure of the cell membrane. No reports of a cell membrane associated with DNA have been reported. I have been studying the preparation of artificial cells and have demonstrated the formation of cells (named DNA crown cells) containing a membrane comprising lipid-DNA. I have prepared DNA crown cells using a known lipid (monolaurin) in vitro and examined whether these cells self-replicate in vivo. Here, I discuss these successful procedures. First, aggregates of sphingosine-DNA were prepared by the addition of adenosine-monolaurin to sphingosine-DNA. Next, DNA crown cells were constructed by the addition of monolaurin to the sphingosine-DNA aggregates in vitro. Finally, I demonstrated that these cells can self-replicate upon incubation in egg-white Keywords: Artificial cells, DNA crown cells, Sphingosine-DNA Adenosine-lipid, Aggregates of sphingosine-DNA, Self-replication was added to the Sph-DNA-A-M mixture. After mixing, the cells were observed as described above. Artificial cell generation using a mixture of Sph-DNA-A-M solution and transplantation To generate artificial cells using the Sph-DNA-A-M mixtures, 90μl of Sph (10 mM) was added to 40μl of DNA (1.7μg/μl). After heating, A-M solution (100μl) was added to the Sph-DNA mixture. A hole was drilled in the shell of an edible white Leghorn egg and 0.25~0.5 ml of the mixture was injected into the egg white (albumin). Each egg was then incubated for 7 days at 37℃, and a portion of each egg white was removed. About 1 ml of egg albumin was added to 10 ml of Dulbecco' Modified Eagles Medium containing 10% bovine serum (DMEM) and cultured for 2 days at 37℃. Aggregates of artificial cells were found at the bottom of the culture medium.
A drop of the aggregates was placed on a glass slide and the integrity of the artificial cells was observed using phase contrast microscopy and fluorescence microscopy. The artificial cells were transplanted by inoculating a sample of egg-white (0.5ml) containing the cells into fresh egg-white weekly. Also, 2 ml of egg-white containing cells was incubated in 10 ml of DMEM and incubated at 37℃ for 2 days. Transplantation was carried out until the fifth generation.

Result and Discussion
Effect of A-M on Sphingosine-DNA To prepare adenosine-lipid compound, the lipid used was monolaurin, based on previous experiments (Inooka, 2016, Ref.8). monolaurin solution was added to adenosine solution. After mixing, ethanol was added, the precipitate was collected and dried, then, the A-M precipitate was resolved in distilled water Sph (90μl,10mM) was added to 40 μl of DNA (1.7μg/μl), heated, then the A-M solution (50μl) was added. One drop of the Sph-DNA-A-M mixture was placed on a glass slide and observed using phase contrast and fluorescence microscopy.
Two types of aggregates were observed. One was mucoid-type, as shown in Fig. 1a, and the other was crystal-type, as shown in Fig. 1b. Typical crystal aggregates are shown in Fig. 1c. Russert light was observed on the edge of the crystal using fluorescence microscopy (Fig. 1d) Fig. 2b, indicating that DNA crown cells had formed. A typical large cell as observed using fluorescence microscopy is shown in Fig. 2c and a typical small cell is shown in Fig. 2d. Russert light is clearly observed on the surfaces of both cells. The shrinkage of large cells may result in the formation of cells of various sizes. Typical crystal-type of aggregates was shown in Fig1c. The sample was stained with ethidium bromide and Russert light was observed on the surfaces of the crystal aggregates under fluorescence microscopy (Fig. 1d), suggesting that the surface contains DNA. Scale bar 50 µm. Fig. 1c and Fig. 1d are the same field of view. c) A typical large particle observed using fluorescence microsc py is shown in Fig. 2c. Russet light was observed on the wall of the cell, indicating that the wall contains DNA. Scale bar 20 µm. d) A typical small cell observed using fluorescence microscopy is shown in Fig. 2d. Russet light was observed on the particle, indicating that particles contain DNA. Scale bar is 20µm.
These large and small cells could be formed by the aggregation of crystal-type and mucoid-type cells. The generation of artificial cells from Sph-DNA-A-M mixtures and transplantation of DNA crown cells in vivo. Sph-DNA-A-M mixtures were prepared as described in Step 3. Each mixture was injected into the white (albumin) of individual eggs. After incubation at 37 0 C for 7 days, 0.5 ml of eggwhite potentially containing artificial cells was inoculated into fresh egg-white weekly for five weeks. One week's incubation is defined as one generation. For each generation, 2 ml of egg-white containing cells was incubated in 10 ml of DMEM and incubated at 37 0 C for 2 days. Aggregates at the bottom of the culture medium were observed. The structural integrity of the artificial cells after 5 generations was assessed by phase contrast microscopy (Fig. 3a) and fluorescence microscopy (Fig. 3b). Three DNA crown cells (arrows) were clearly observed, indicating that DNA crown cells replicated in egg-white.
Thus, I have succeed in preparing DNA crown cells using the purified reagents sphingosine, DNA, adenosine and monolaurin. I have also demonstrated ; Ref.7) that artificial cells are generated using Sph-DNA and nucleosides, including uridine. Therefore, mixtures prepared with combinations of nucleosides and monolaurin may aggregate Sph-DNA. Moreover, compounds prepared using combinations of nucleosides and lipids related to monolaurin may also form aggregates of Sph-DNA and thus construct DNA crown cells. Here, I used DNA from Escherichia coli. These cells can be prepared using DNA from other sources as well.
This means that various DNA crown cells consisting of different components could be prepared by combining nucleosides, lipids and DNA. The current method for preparing DNA crown cells can be performed easily. Cells whose membrane consists of DNAlipid have not previously been reported. This method is readily accessible to anyone and may provide new findings in the life sciences, especially regarding the mechanisms underlying virus replication.