Technical and Macromolecular Chemistry, Paderborn University, Warburger Str. 100, 33098 Paderborn, Germany.
Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
Molecules. 2020 Nov 3;25(21):5099. doi: 10.3390/molecules25215099.
Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA-protein nanostructures may find applications in the crystallization of DNA-protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.
不动 Holliday 连接不仅代表了结构 DNA 纳米技术的最基本构建块，而且对于体外研究遗传重组和表观遗传学也具有重要意义。在这里，我们在单链退火蛋白 Redβ的帮助下，对室温下不动 Holliday 连接的组装进行了详细研究。单个 DNA 单链最初用蛋白单体覆盖，然后杂交形成刚性的钝端四臂连接。我们研究了不同 DNA/蛋白比以及不同 DNA 序列长度对这种方法的效率。此外，我们还评估了 Redβ将粘性末端修饰的 Holliday 连接点退火成分级组装的潜力。我们展示了 Redβ介导的 Holliday 连接子二聚体、多聚体和几微米大小的扩展网络的退火。虽然这些杂交 DNA-蛋白质纳米结构可能在 DNA-蛋白质复合物的结晶中找到应用，但我们的工作表明 Redβ 在温和的反应条件下辅助功能性 DNA 纳米结构合成方面具有巨大的潜力。
