Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale
Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale
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DOI：
10.1021/acscentsci.9b00174
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发表时间：
2019-05-22
影响因子：
18.2
通讯作者：
Sleiman, Hanadi F.
中科院分区：
文献类型：
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作者：
Lacroix, Aurelie;Vengut-Climent, Empar;Sleiman, Hanadi F.
DNA寡核苷酸和纳米结构的荧光染料标记是最常用的技术之一，以跟踪它们的命运和细胞内的细胞定位。在这里，我们报告说，细胞内的荧光，甚至FRET信号，不能与细胞摄取完整的DNA结构。活细胞成像显示高共定位的花青标记的DNA寡核苷酸和磷酸化的小分子花青染料，从这些DNA化合物的降解产物之一的纳米结构。细胞内外链的核酸酶降解导致误导性的细胞内荧光信号。降解产物（磷酸化染料）的荧光使信号饱和。为了验证我们的假设，我们合成了一系列DNA结构，包括Cy3和Cy5标记的DNA立方体和DNA四面体，以及对核酸酶具有不同稳定性的寡核苷酸。所有细胞摄取后在线粒体内产生荧光信号，并与游离磷酸化染料对照强烈共定位。动力学实验表明，稳定的DNA结构的摄取被延迟。我们还研究了影响荧光数据的几个参数：DNA链的稳定性，固定方法，可以洗掉信号，染料的DNA链上的位置，和FRET实验的设计。DNA纳米结构由于其生物相容性、可编程性和易于合成而在生物医学应用和生物技术方面具有巨大的潜力。然而，很少有成功的DNA机器在体内的例子已经报道。我们相信这一贡献可以作为指导，以设计更好的细胞摄取实验时，使用荧光染料，以进一步推动生物学的发展，和DNA纳米结构的应用。
Fluorescent dye labeling of DNA oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells. Here, we report that intracellular fluorescence, and even FRET signals, cannot be correlated with the cellular uptake of intact DNA structures. Live cell imaging revealed high colocalization of cyanine-labeled DNA oligos and nanostructures with phosphorylated small-molecule cyanine dyes, one of the degradation products from these DNA compounds. Nuclease degradation of the strands outside and inside the cell results in a misleading intracellular fluorescent signal. The signal is saturated by the fluorescence of the degradation product (phosphorylated dye). To test our hypothesis, we synthesized a range of DNA structures, including Cy3- and Cy5-labeled DNA cubes and DNA tetrahedra, and oligonucleotides with different stabilities toward nucleases. All give fluorescence signals within the mitochondria after cellular uptake and strongly colocalize with a free phosphorylated dye control. Kinetics experiments revealed that uptake of stable DNA structures is delayed. We also studied several parameters influencing fluorescent data: stability of the DNA strand, fixation methods that can wash away the signal, position of the dye on the DNA strand, and design of FRET experiments. DNA nanostructures hold tremendous potential for biomedical applications and biotechnology because of their biocompatibility, programmability, and easy synthesis. However, few examples of successful DNA machines in vivo have been reported. We believe this contribution can be used as a guide to design better cellular uptake experiments when using fluorescent dyes, in order to further propel the biological development, and application of DNA nanostructures.