Department of Molecular Biology and Plant Virology, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany.
Institute of Organic Chemistry, University of Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
Nanoscale. 2018 Apr 5;10(14):6496-6510. doi: 10.1039/C7NR07743B.
By combining both chemical and enzymatic ligation with procedures guiding the self-assembly of nanotubular tobacco mosaic virus (TMV)-like particles (TLPs), novel nucleoprotein structures based on DNA-terminated branching elements, RNA scaffolds and TMV coat protein (CP) are made accessible. Tetrahedral tetrakis(hydroxybiphenyl)adamantane cores with four 5'-phosphorylated dinucleotide arms were coupled to DNA linkers by chemical ligation. The resulting three-dimensional (3D) branching elements were enzymatically ligated to the 3' termini of RNA scaffolds either prior to or after the RNAs' incorporation into TLPs. Thus, architectures with interconnected nanotube domains in two different length classes were generated, each containing 70 CP subunits per 10 nm length. Short TMV origin-of-assembly-containing RNA scaffolds ligated to the DNA allowed the growth of protein-coated 34 nm tubes on the terminal RNA strands in situ. Alternatively, 290 nm pre-fabricated tubes with accessible RNA 3' termini, attained by DNA blocking elements hybridized to the RNAs, were ligated with the branched cores. Both approaches resulted in four-armed nanoobjects, demonstrating a so far unique combination of organic synthesis of branching elements, enzymatic modifications, nucleic acid-based scaffolding and RNA-guided and DNA-controlled assembly of tubular RNA-encapsidating protein domains, yielding a novel class of 3D nucleoprotein architectures with polyvalent protein elements. In the long term, the production route might give rise to supramolecular systems with complex functionalities, installed via the orthogonal coupling of effector molecules to TLP domains.
通过将化学和酶连接与指导管状烟草花叶病毒（TMV）样颗粒（TLP）自组装的程序相结合，基于 DNA 末端分支元件、RNA 支架和 TMV 外壳蛋白（CP）的新型核蛋白结构得以实现。具有四个 5'-磷酸二核苷酸臂的四面四羟联苯金刚烷核心通过化学连接与 DNA 接头偶联。所得的三维（3D）分支元件通过酶连接到 RNA 支架的 3'末端，要么在将 RNA 掺入 TLP 之前，要么在之后。因此，生成了两种不同长度类别的具有互连纳米管结构域的结构，每个结构域包含每个 10nm 长度 70 个 CP 亚基。与 DNA 连接的短 TMV 组装起始 RNA 支架允许在原位在末端 RNA 链上生长蛋白质涂层的 34nm 管。或者，通过与 RNA 杂交的 DNA 阻塞元件获得的具有可访问的 RNA 3'末端的 290nm 预制管与分支核心连接。这两种方法都得到了四臂纳米物体，证明了分支元件的有机合成、酶修饰、基于核酸的支架以及 RNA 引导和 DNA 控制的管状 RNA 封装蛋白结构域的组装的独特组合，产生了一类具有多价蛋白元件的新型 3D 核蛋白结构。从长远来看，该生产路线可能会产生具有复杂功能的超分子系统，通过效应分子与 TLP 结构域的正交偶联来安装。
