一种采用主动靶向策略的DNA四面体输药载体可促进稳定的药物包封和刺激响应型随需释放，构建不同药物输送需求的通用平台。由于其极佳的生物兼容性、可编程性和显著的细胞和组织渗透性，四面体DNA纳米结构（TDN）已经在各种生物活性分子的输送中证明了其价值。我们先前在早期规程中将其描述为静态多功能复合物。然而，在复杂的生物条件下，静态结构和被动靶向行为可能会引入离靶效应。因此，在这个协议扩展中，我们提出了TDN输送载体的重大更新，使其可以使用主动靶向策略进行刺激敏感的构象变化和现场货物释放，从而避免了其他基于DNA的输送载体的复杂和耗时的制造过程和未确定的细胞穿透能力的缺点。在精美的TDN尺寸设计基础上，采用一步退火方法制备Tiamat设计的TDN外骨骼。然后，动态DNA装置的设计可以基于目标和环境刺激，包括基于DNA链杂交的和pH敏感的DNA装置，并使用聚丙烯酰胺和琼脂糖凝胶电泳或荧光染料修饰来达到链长和不匹配的仔细滴定。最后，设计货物加载策略，包括站点和准确的滴定和货物包封验证。动态结构在体内外的抗肿瘤和抗炎治疗中显示出有前途的靶向性和有效性。实验室中的组装和表征需要约5天，生物稳定性和生物应用的验证时间取决于用途。©2023 Spring Nature Limited.

An active targeting strategy-enabled DNA tetrahedron delivery vehicle could facilitate stable drug encapsulation and stimuli-responsive on-demand release, building a universal platform for different drug delivery requirements. Owing to the excellent biocompatible nature, programmability and remarkable cell and tissue permeability, the tetrahedral DNA nanostructure (TDN) has proven its value in the delivery of various bioactive molecules. We previously described this as a static multifunctional complex in our earlier protocol. However, static structures and passive targeting behavior might introduce off-target effects under complicated biological conditions. Therefore, in this Protocol Extension, we present a major update of the TDN delivery vehicle enabling an active targeting strategy to be used for stimuli-sensitive conformation changes and on-site cargo release, which could avoid drawbacks, including complex and time-consuming fabrication processes and undetermined cell penetration ability of other DNA-based delivery vehicles. Upon exquisite design of TDN size based on cargo type, one-pot annealing is applied to fabricate the Tiamat-designed TDN exoskeleton. Then the design of the dynamic DNA apparatus can be based on the target and environmental stimuli, including DNA strand hybridization-based and pH-sensitive DNA apparatus, and careful titration of strand lengths and mismatches is achieved using polyacrylamide and agarose gel electrophoresis, or fluorophore modifications. Finally, cargo loading strategies are designed, including site and stand titration and cargo encapsulation verification. The dynamic structures show promising targetability and effectiveness in antitumor and anti-inflammatory treatment in vitro and in vivo. Assembly and characterization in the lab takes ~5 d, and the timing for the verification of biostability and biological applications depends on the uses.© 2023. Springer Nature Limited.