College of Chemistry and Chemical Engineering, the Hunan Provincial Key Laboratory of Water Environment and Agriculture Product Safety, Central South University, Changsha 410083, Hunan, China.
College of Life Science, Central South University, Changsha 410083, Hunan, China.
Anal Chem. 2024 Jan 9;96(1):564-571. doi: 10.1021/acs.analchem.3c04837. Epub 2023 Dec 19.
DNA nanostructure-based signal amplifiers offer new tools for imaging intracellular miRNA. However, the inadequate kinetics and susceptibility to enzymatic hydrolysis of these amplifiers, combined with a deficient cofactor concentration within the intracellular environment, significantly undermine their operational efficiency. In this study, we address these challenges by encapsulating a localized target strand displacement assembly (L-SD) and a toehold-exchange endogenous-powered component (R-mRNA) within a framework nucleic acid (FNA) structure─20 bp cubic DNA nanocage (termed RL-cube). This design enables the construction of an endogenous-powered and spatial-confinement DNA nanomachine for ratiometric fluorescence imaging of intracellular miRNA Let-7a. The R-mRNA is designed to be specifically triggered by glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an abundant cellular enzyme, and concurrently releases a component that can recycle the target Let-7a. Meanwhile, L-SD reacts with Let-7a to release a stem-loop beacon, generating a FRET signal. The spatial confinement provided by the framework, combined with the ample intracellular supply of GAPDH, imparts remarkable sensitivity (7.57 pM), selectivity, stability, biocompatibility, and attractive dynamic performance (2240-fold local concentration, approximately four times reaction rate, and a response time of approximately 7 min) to the nanomachine-based biosensor. Consequently, this study introduces a potent sensing approach for detecting nucleic acid biomarkers with significant potential for application in clinical diagnostics and therapeutics.
基于 DNA 纳米结构的信号放大器为细胞内 miRNA 的成像提供了新的工具。然而，这些放大器的动力学不足和对酶水解的敏感性，以及细胞内环境中辅酶浓度的不足，极大地降低了它们的运行效率。在这项研究中，我们通过将局部目标链置换组装（L-SD）和一个适体交换内源性动力组件（R-mRNA）封装在一个框架核酸（FNA）结构——20bp 立方 DNA 纳米笼（称为 RL-cube）中，来解决这些挑战。该设计能够构建一个内源性动力和空间约束的 DNA 纳米机器，用于细胞内 miRNA Let-7a 的比率荧光成像。R-mRNA 被设计为专门被甘油醛 3-磷酸脱氢酶（GAPDH）触发，GAPDH 是一种丰富的细胞酶，同时释放一个可以循环利用目标 Let-7a 的组件。同时，L-SD 与 Let-7a 反应，释放出一个茎环信标，产生 FRET 信号。框架提供的空间约束，加上细胞内充足的 GAPDH 供应，赋予了纳米机器生物传感器显著的灵敏度（7.57pM）、选择性、稳定性、生物相容性和有吸引力的动态性能（2240 倍的局部浓度、大约四倍的反应速率和大约 7 分钟的响应时间）。因此，本研究提出了一种强大的检测核酸生物标志物的方法，具有在临床诊断和治疗中应用的巨大潜力。
