State Key Laboratory of Chemeo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China.
J Am Chem Soc. 2015 Jun 3;137(21):6829-36. doi: 10.1021/jacs.5b01778. Epub 2015 May 20.
Efficient approaches for intracellular delivery of nucleic acid reagents to achieve sensitive detection and regulation of gene and protein expressions are essential for chemistry and biology. We develop a novel electrostatic DNA nanoassembly that, for the first time, realizes hybridization chain reaction (HCR), a target-initiated alternating hybridization reaction between two hairpin probes, for signal amplification in living cells. The DNA nanoassembly has a designed structure with a core gold nanoparticle, a cationic peptide interlayer, and an electrostatically assembled outer layer of fluorophore-labeled hairpin DNA probes. It is shown to have high efficiency for cellular delivery of DNA probes via a unique endocytosis-independent mechanism that confers a significant advantage of overcoming endosomal entrapment. Moreover, electrostatic assembly of DNA probes enables target-initialized release of the probes from the nanoassembly via HCR. This intracellular HCR offers efficient signal amplification and enables ultrasensitive fluorescence activation imaging of mRNA expression with a picomolar detection limit. The results imply that the developed nanoassembly may provide an invaluable platform in low-abundance biomarker discovery and regulation for cell biology and theranostics.
高效的方法将核酸试剂递送到细胞内，以实现对基因和蛋白质表达的敏感检测和调控，这对化学和生物学来说是至关重要的。我们开发了一种新型的静电 DNA 纳米组装体，它首次实现了杂交链式反应（HCR），这是一种在活细胞中两种发夹探针之间进行的目标起始的交替杂交反应，用于信号放大。该 DNA 纳米组装体具有一个设计好的结构，包括一个核心金纳米颗粒、一个阳离子肽中间层和一个静电组装的带有荧光标记发夹 DNA 探针的外层。研究表明，它通过一种独特的非内吞作用机制高效地将 DNA 探针递送到细胞内，这一机制赋予了克服内涵体捕获的显著优势。此外，DNA 探针的静电组装使得探针能够通过 HCR 从纳米组装体中被目标物触发释放。这种细胞内 HCR 提供了高效的信号放大，并能够以皮摩尔检测限对 mRNA 表达进行超灵敏的荧光激活成像。这些结果表明，所开发的纳米组装体可能为细胞生物学和治疗学中的低丰度生物标志物发现和调控提供一个非常有价值的平台。
