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

展示巨型囊泡和 DNA 同步自我复制动力学的原始细胞模型

基本信息

批准号：
17350066
负责人：
SUGAWARA Tadashi
金额：
$ 8.92万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

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

1.以10-（对甲酰基苯氧基）癸醇和十二醇为原料，采用亚酰胺法合成了一种新型的阴离子极性头部为杂磷酸二酯的膜分子，产率为62%。这种阴离子膜分子在含15%甲醇的磷酸盐缓冲溶液中形成了几乎单层的巨囊泡。此外，当加入膜前体时，阴离子囊泡表现出自我繁殖动力学（The 88 th Annual Meeting of Chemical Society of Japan，3L 3 -17）。在由POPC构成的巨囊泡中的聚合酶链反应（PCR）使用PCR技术在POPC（棕榈酰-油酰-磷脂酰胆碱）的巨囊泡（GV）内进行由可表达GFP的1229 bp构成的模板DNA的复制。20次热循环后，通过荧光显微镜观察，DNA双链上吸收的SYBR绿色I发出的荧光变得清晰可见（平均增加两个数量级） ...更多信息示波器。通过PAGE（聚丙烯酰胺凝胶电泳）确认模板DNA的精确复制。流式细胞仪分析显示，GVs内DNA的有效复制率约为10%（The 88 th Annual Meeting of Chemical Society of Japan，3C 6 -25）.为了上述目的，制备了由DNA寡聚体（15聚体）、间隔物和胆固醇组成的缀合物分子。DNA寡聚体作为引物与GV内的模板DNA（100 mer）杂交，胆固醇作为锚将缀合物分子与囊泡膜固定。用荧光探针标记引物，在荧光显微镜下直接观察模板DNA在囊泡膜内表面的复制过程，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学之间可能的耦合，这对于产生人工细胞模型至关重要（Soft Matter，2005）。少