Upconversion Nanoparticle-Based FRET System for Study of siRNA in Live Cells
Upconversion Nanoparticle-Based FRET System for Study of siRNA in Live Cells
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DOI：
10.1021/la904011q
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发表时间：
2010-05-04
期刊：
影响因子：
3.9
通讯作者：
Zhang, Yong
中科院分区：
文献类型：
--
作者：
Jiang, Shan;Zhang, Yong
在纳米粒子将小干扰RNA（siRNA）递送至细胞后，对其细胞内命运的研究对于开发更高效的siRNA转染方法具有重要意义。建立了一种基于荧光共振能量转移（FRET）的方法，该方法以上转换荧光纳米粒子（UCN）作为能量供体，用于研究活细胞中siRNA的细胞内释放和生物稳定性。制备了UCN/siRNA - BOBO3复合物，其中BOBO - 3标记的siRNAs附着在氨基修饰的二氧化硅/NaYF4:Yb,Er UCN表面。在近红外（NIR）激光激发下，能量从UCN供体转移到BOBO - 3受体。FRET效率被确定为一个可靠的参数，用于追踪siRNA在磷酸盐缓冲盐水（PBS）和活细胞中的释放和生物稳定性。细胞内FRET分析表明，在24小时内siRNA逐渐释放到细胞中，这通过共聚焦显微镜共定位测量得到了证实。这种简单而灵敏的上转换FRET技术的应用可以获得关于siRNA细胞内命运的实时信息，并为生物分子的体外甚至体内检测提供了良好的前景。
Investigation or the intracellular fate of small interference RNA (si RNA), after their delivery into cells by nanoparticles, is of great interest to the development of more efficient methods for transfection of si RNA. The fluorescence resonance energy transfer (FRET) based method using upconversion fluorescent nanopartieles (UCN) as energy donor is established to study intracellular release and biostability of si RNA in live cells. The UCN/siRNA-BOBO3 complex is prepared where BOBO-3-stained siRNAs are attached to the surface of amino-group-modified silica/NaYF4:Yb,Er UCN. The energy is transferred from the UCN donor to the BOBO-3 acceptor under excitation of a near-infrared (NIR) laser. The FRET efficiency is established as a reliable parameter to follow the release and biostability of siRNA in phosphate buffered saline (PBS) and live cells. Intracellular FRET analysis shows that siRNA is gradually released into cells for a duration of 24 h, which is confirmed by confocal microscopy colocalization measurements. The application of this straightforward and sensitive upconversion FRET technique can gain real-time information on intracellular fate of si RNA and provide a bright outlook for in vitro and even in vivo detection of biological molecules.