响应型DNA折纸递送mRNA

Responsive DNA origami for mRNA delivery

摘要:

mRNA技术已成为疫苗、基因治疗与再生医学等领域的重要工具。然而，mRNA在生理环境中易被核糖核酸酶(RNase)快速降解，且分子量大、带负电导致其难以跨膜进入细胞；现有递送体系面临血清稳定性不足、细胞摄取效率有限以及胞内可控释放机制缺乏等关键瓶颈，亟需发展兼具稳定保护与可控释放能力的新型递送系统。为了构建该系统，本研究采用 DNA 折纸技术构建了一种筒状(barrel) DNA纳米结构，并通过琼脂糖凝胶电泳、原子力显微镜(atomic force microscope, AFM)和透射电镜(transmission electron microscope, TEM)验证其结构完整性；在10%胎牛血清(fetal bovine serum, FBS)条件下验证mRNA-折纸复合物结构的稳定性，并评估其在HT29细胞中的摄取行为，基于该平台，进一步开发刺激响应型释放策略，引入紫外光可激活的PC linker，实现光控触发释放，从而实现mRNA的响应性翻译表达。所构建的光控释放系统能够在特定刺激下实现可编程的胞内解离与释放，将载体结构优化与刺激响应机制相结合，形成一套完整的智能mRNA递送方案。本研究为构建具备高稳定性、高摄取效率与可控释放能力的核酸递送系统提供了新的设计思路与实验范例。

Abstract:

mRNA technology has emerged as a crucial tool in fields including vaccinology, gene therapy, and regenerative medicine. However, mRNA is susceptible to rapid degradation by ribonucleases (RNases) in physiological environments, and its large molecular weight and negative charge hinder its transmembrane delivery into cells. Existing delivery systems face key bottlenecks such as insufficient serum stability, limited cellular uptake efficiency, and a lack of controlled intracellular release mechanisms. Consequently, there is an urgent need to develop novel delivery platforms that offer both robust protective capabilities and precisely controlled release. In this study, a barrel-shaped DNA nanostructure was constructed using DNA origami technology, and its structural integrity was verified using agarose gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). The stability of the mRNA-origami complex was further assessed under 10% fetal bovine serum (FBS) conditions, and its cellular uptake behavior was evaluated in HT29 cells. Building on this platform, a stimulus-responsive release strategy was further developed by incorporating an ultraviolet-activatable PC linker, enabling photo-controlled release and thereby achieving responsive mRNA translation expression. The constructed photo-controlled release system allows programmable intracellular dissociation and release upon specific stimuli. By combining carrier structure optimization with a stimulus-responsive mechanism, a comprehensive intelligent mRNA delivery strategy was established. This study provides novel design concepts and experimental paradigms for constructing nucleic acid delivery systems with high stability, efficient cellular uptake, and controllable release capability.