Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
Department of Microbiology, Harvard Medical School, Boston, MA, USA.
Nat Commun. 2023 Jan 24;14(1):382. doi: 10.1038/s41467-023-36156-1.
Hybrid RNA:DNA origami, in which a long RNA scaffold strand folds into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of three-dimensional wireframe RNA-scaffolded origami rendered as polyhedra composed of dual-duplex edges. We computationally design, fabricate, and characterize tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds. We characterize secondary and tertiary structures using dimethyl sulfate mutational profiling and cryo-electron microscopy, revealing insight into both global and local, base-level structures of origami. Our top-down sequence design strategy enables the use of long RNAs as functional scaffolds for complex wireframe origami.
杂交 RNA:DNA 折纸术，其中长 RNA 支架链通过与互补 DNA 寡核苷酸的热退火折叠成目标纳米结构，尽管其在 mRNA 的生物医学递送上具有独特的潜力、长 RNA 的三级结构表征和人工核酶的制造方面具有独特的潜力，但仅在有限的程度上进行了探索。在这里，我们研究了作为由双双链边缘组成的多面体呈现的三维线框 RNA 支架折纸的设计原则。我们通过计算设计、制造和表征了由 EGFP 编码信使 RNA 和德布鲁因序列折叠而成的四面体、用 M13 转录 RNA 折叠的八面体以及用 23S 核糖体 RNA 折叠的八面体和五边形双锥，展示了用不同的 RNA 支架制造不同的多面体形状的能力具有独特的结构和功能。我们使用硫酸二甲酯突变分析和冷冻电子显微镜来表征二级和三级结构，揭示了折纸术的全局和局部、碱基水平结构的洞察力。我们的自上而下的序列设计策略使长 RNA 能够用作复杂线框折纸术的功能支架。
