Development of Clonable Electron-Dense Labels for Electron Tomography

我们正在研究开发可克隆的电子密度标记的可行性，这种标记可以在通过电子断层扫描获得的亚细胞细胞器的三维重建中可视化。目前，在确定哪些感兴趣的蛋白质对应于重建中的特定特征时，电子断层扫描往往受到不确定性的限制。可克隆的标记可能通过类似于光学显微镜中的绿色荧光蛋白来定位这些蛋白质。细菌铁蛋白是一种铁储存蛋白，它在中空核心积累了致密的氧化铁纳米颗粒，是一个很有希望的候选蛋白。将诺氏李斯特菌的铁蛋白基因克隆到大肠杆菌中，使插入的DNA编码由柔性连接序列连接的铁蛋白亚基簇。合成并克隆了含有2、4、8和12个连接铁蛋白亚基的载体。目前，我们正在优化体内铁蛋白在大肠杆菌中的载铁条件。 表达可克隆标记的另一种方法涉及开发功能RNA标记。在布鲁斯·夏皮罗博士(NCI，Frederick)的合作下，编码纳米环和纳米管RNA的DNA已经克隆到大肠杆菌中。为了表达这种结构，已经开发了一种T7驱动的表达载体，用于修饰的Naning版本。为了获得自组装所需的RNA，纳米环RNA的两侧有两个核酶(Hammerhead ribozyme和Varkud sat ribozyme)。在体内被这两种核酶切割后，产生了纳米环单体，它可以自组装成环和管。目前，该系统的表达和组装条件正在优化。

We are investigating the feasibility of developing clonable electron-dense labels that can be visualized in three-dimensional reconstructions of subcellular organelles obtained by electron tomography. Currently, electron tomography is often limited by uncertainty in determining which proteins of interest correspond to specific features in the reconstruction. Clonable labels could potentially localize those proteins, by analogy to green fluorescent protein in optical microscopy. Bacterial ferritin, an iron storage protein that accumulates a dense iron oxide nanoparticle in its hollow core, is a promising candidate. A modified ferritin gene from Listeria innocua was cloned into Escherichia coli so that the inserted DNA coded for clusters of ferritin subunits connected by flexible linker sequences. Constructs with 2,4,8 and 12 linked ferritin subunits were synthesized and cloned. Currently we are optimizing conditions for the in vivo iron-loading of ferritin into E. coli. Another approach to expressing clonable markers involves the development of functional RNA labels. In collaboration with Dr. Bruce Shapiro (NCI, Frederick), DNA coding for Nanoring and Nanotube RNA has been cloned into E. coli. In order to express this structure, a T7-driven expression vector for a modified version of the Nanoring has been developed. To obtain the RNA required for self-assembly the Nanoring RNA is flanked by two ribozymes (Hammerhead ribozyme and Varkud satellite ribozyme). After in vivo cleavage by these two ribozymes the Nanoring monomer is generated, which can self-assemble into rings and tubes. Currently expression and assembly conditions of this system are being optimized.