Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a “plug-and-play” rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

中文翻译：

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用于可编程多功能性和延长循环的白蛋白-霍利迪连接生物分子模块化设计

需要将长循环、位点和细胞特异性参与等组合特性纳入先进药物输送系统的设计中，以最大限度地提高药物有效负载功效。这项工作引入了一种四链寡核苷酸霍利迪连接点 (HJ) 基序，其功能部分与重组人白蛋白 (rHA) 共价缀合，形成“即插即用”的 rHA-HJ 多功能生物分子组装体，具有延长的循环能力。电泳凝胶迁移测定显示了各个高性能液相色谱纯化模块的成功功能化和纯度以及 rHA-HJ 构建体的有效组装。包含表皮生长因子受体 (EGFR) 靶向纳米抗体模块有助于与表达 EGFR 的细胞特异性结合，通过流式细胞术分析确定，与不包含纳米抗体的组件相比，荧光强度增加约 150 倍。细胞回收测定证明了保留的白蛋白-新生儿 Fc 受体 (FcRn) 结合亲和力以及伴随的 FcRn 驱动的细胞回收。这意味着双转基因人源化 FcRn/白蛋白小鼠的循环半衰期延长了 4 倍（rHA-HJ 和 HJ 分别为 2.2 和 0.55 小时）。这项工作介绍了一种新型生物分子白蛋白-核酸结构，由于其模块化设计，具有延长的循环半衰期和可编程多功能性。