Gold-tagged RNA - A probe for macromolecular assemblies
Gold-tagged RNA - A probe for macromolecular assemblies
复制标题
DOI：
10.1006/jsbi.1999.4134
复制
发表时间：
1999-09-01
影响因子：
3
通讯作者：
Sperling, J
中科院分区：
文献类型：
--
作者：
Medalia, O;Heim, M;Sperling, J
核糖核酸（rna）在许多基本生命过程中起着关键作用。这些聚合物通常与蛋白质以分子质量可达数百万道尔顿的极大颗粒（如核糖体、剪接体和病毒）络合在一起。对这类rna -蛋白复合物的结构研究将有助于阐明它们的作用方式。对于许多大分子组件的结构分析，电子显微镜（EM）起着重要的作用。然而，通过EM定位生物复合体中的RNA还不是一项直截了当的工作。在这里，我们描述了一种方法，共价标记的RNA分子与金簇，从而使其直接可视化的显微镜方法。我们的策略包括在体外转录携带游离巯基的rna，使用三磷酸核糖核苷类似物在其杂环上含有末端巯基的取代基。这个合成之后，金簇通过一个雄性胺基偶联到硫代转录物上。通过透射电子显微镜对这些金标记RNA的可视化显示，直径为1-2纳米的金簇以几乎规则的距离排列在一条假想曲线上，可能与RNA链相对应。原子力显微镜显示的RNA链图像证实了这一结论，其中可以清楚地看到球形结构，其高度与金簇的直径相对应。这项研究展示了用金簇共价标记的核酸用于蛋白质- rna复合物的结构表征的潜在用途，(C) 1999年学术出版社。
Ribonucleic acids (RNAs) play a key role in many fundamental life processes. These polymers are often found complexed with proteins in extremely large particles whose molecular mass may reach several millions of daltons (e.g. ribosomes, spliceosomes, and viruses). Structural studies of such RNA-protein complexes should help elucidate their mode of action. For the structural analyses of many macromolecular assemblies, electron microscopy (EM) has served an instrumental role. However, localization by EM of RNA within biological complexes is not yet a straightforward undertaking. Here we describe a methodology for the covalent tagging of RNA molecules with gold clusters, thereby enabling their direct visualization by microscopical methods. Our strategy involves transcription in vitro of RNAs that carry free thiol groups, using ribonucleoside triphosphate analogs containing a substituent with a terminal thiol group on their heterocyclic ring. This synthesis is followed by coupling of gold clusters to the thiolated transcript through a maleimido group. Visualization of such gold-tagged RNAs by transmission electron microscopy showed spots of gold clusters, with a diameter of 1-2 nm, arranged at nearly regular distances on an imaginary curve that presumably corresponds to the RNA chain. This assignment was corroborated by atomic force microscopy that exhibited images of RNA chains in which knoblike structures, whose height corresponds to the diameter of the gold clusters, were clearly seen. This study demonstrates the potential use of nucleic acids that are covalently labeled with gold clusters for the structural characterization of protein-RNA complexes, (C) 1999 Academic Press.