Proto-cell Model Exhibiting Synchronized Self-replication Dynamics of Giant Vesicles and DNA

1.由阴离子膜分子组成的自复制巨囊泡以10-(对甲酰基苯氧基)十二醇为原料，采用酰胺法制备了一种新型的以杂磷酸二酯为阴离子极性头的膜分子，产率为62%。这种阴离子膜分子在含有15%甲醇的磷酸盐缓冲溶液中形成了几乎单层的巨大囊泡。此外，当加入膜前体时，阴离子囊泡表现出自我复制的动力学(日本化学会第88届年会，3L3-17)。利用聚合酶链式反应(PCR)技术，在POPC(Palmitoyl-Oleoyl-Patidyl Choline，棕榈酰-油酰-磷脂酰胆碱)的巨大囊泡内复制可表达绿色荧光蛋白的模板DNA，该模板DNA由1229个DNA组成。经过20个热循环后，SYBR Green I吸收在DNA-双链上的荧光通过荧光MICR…变得清晰可见(平均增加两个数量级更多的观察镜。用PAGE(聚丙烯酰胺凝胶电泳法)证实了模板DNA的准确复制。流式细胞仪分析显示，DNA在GVS中的有效复制率约为10%(日本化学会第88届年会，3C6-25)。DNA自复制动力学和巨泡自复制动力学的耦合为了达到上述目的，我们制备了由DNA低聚物(15聚体)、间隔基和胆固醇组成的偶联分子。DNA寡聚体作为与GV内模板DNA(100聚体)杂交的引物，而胆固醇作为锚定，将结合分子与囊泡膜固定。用带有荧光探针的引物在荧光显微镜下直接观察到模板DNA在囊泡膜内表面的互补复制过程，并通过PAGE分析证实了模板DNA的复制。这种方法保证了两种动力之间可能的耦合，这对于产生人造细胞模型至关重要(Soft Matter，2005)。较少

1. Self-reproducing giant vesicles composed of anionic membrane molecules A novel membrane molecule with an anionic polar head comprised of hetero-phosphate diester was prepared from 10-(p-formylphenoxy) decanol and dodecanol through an amidite method in a 62 % yield. This anionic membrane molecule constructed nearly uni-lamella giant vesicles in a phosphate buffer solution containing 15% methanol. Moreover the anionic vesicles exhibited self-reproduction dynamics when a membrane-precursor was added (The 88th Annual Meeting of Chemical Society of Japan, 3L3-17).2. Polymerase chain reaction (PCR) in giant vesicles composed of POPC Replication of template DNA consisting of 1229 by that can express GFP was performed inside giant vesicles (GV) of POPC (palmitoyl-oleoyl-phosphatidyl choline) using a PCR technique. After 20 thermal cycles, the fluorescence emitted from SYBR Green I absorbed on the DNA-double strands became clearly visible (two orders increase in average) by fluorescence micr … More oscope. The exact replication of the template DNA was confirmed by PAGE (polyacrylamide gel electrophoresis). Flow-cytometric analysis revealed that ratio of the effective replication of DNA inside GVs was ca 10 % (The 88th Annual Meeting of Chemical Society of Japan, 3C6-25).3. Coupling of dynamics of self-replication of DNA and self-reproduction of giant vesicles For the purpose titled as above, a conjugate molecule consisting of a DNA oligomer (15mer), a spacer, and cholesterol was prepared. The DNA oligomer serves as a primer hybridizing with a template DNA (100mer) inside GV, and cholesterol works as, an anchor to fix the conjugate molecule with the vesicular membrane. The progress of the complimentary replication of the template DNA on the inner surface of vesicular membrane was directly observed by a fluorescent microscope using a primer tagged with a fluorescence-probe, and the replication of the template DNA was confirmed by PAGE analysis. This approach guarantees the possible coupling between the two dynamics, which is crucial for giving rise to an artificial cell model (Soft Matter, 2005). Less