Toehold-Mediated Selective Assembly of Compact Discrete DNA Nanostructures

Production of small discrete DNA nanostructures containing covalent junctions requires reliable methods for the synthesis and assembly of branched oligodeoxynucleotide (ODN) conjugates. This study reports an approach for self-assembly of hard-to-obtain primitive discrete DNA nanostructures“nanoethylenes”, dimers formed by double-stranded oligonucleotides using V-shaped furcate blocks. We scaled up the synthesis of V-shaped oligonucleotide conjugates using pentaerythritol-based diazide and alkyne-modified oligonucleotides using copper­(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and optimized the conditions for “nanoethylene” formation. Next, we designed nanoethylene-based “nanomonomers” containing pendant adapters. They demonstrated smooth and high-yield spontaneous conversion into the smallest cyclic product, DNA tetragon aka “nano-methylcyclobutane”. Formation of DNA nanostructures was confirmed using native polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM) and additionally studied by molecular modeling. The proposed facile approach to discrete DNA nanostructures using precise adapter-directed association expands the toolkit for the realm of DNA origami.制备带有共价连接位点的小型离散DNA纳米结构，需要可靠的支链寡脱氧核苷酸（oligodeoxynucleotide, ODN）缀合物合成与组装方法。本研究报道了一种针对难以获取的原始离散DNA纳米结构的自组装策略——“纳米乙烯（nanoethylenes）”，即由双链寡核苷酸通过V形分叉模块形成的二聚体。我们采用基于季戊四醇的双叠氮化合物与炔基修饰寡核苷酸，通过铜(I)催化叠氮-炔环加成（copper(I)-catalyzed azide-alkyne cycloaddition, CuAAC）放大了V形寡核苷酸缀合物的合成工艺，并优化了“纳米乙烯”的形成条件。随后，我们设计了带有悬挂式适配器的基于纳米乙烯的“纳米单体”，该单体可顺利实现高产率的自发转化，生成最小的环状产物——DNA四元环，又称“纳米甲基环丁烷（nano-methylcyclobutane）”。通过非变性聚丙烯酰胺凝胶电泳（native polyacrylamide gel electrophoresis, PAGE）与原子力显微镜（atomic force microscopy, AFM）验证了DNA纳米结构的形成，并通过分子建模开展了补充研究。本研究提出的借助精准适配器导向缔合制备离散DNA纳米结构的简便策略，拓展了DNA折纸术（DNA origami）的研究工具库。