Sato Takaya, Sato Yusuke, Iwai Kenta, Kuge Shusuke, Teramae Norio, Nishizawa Seiichi
Department of Chemistry, Graduate School of Science, Tohoku University.
Anal Sci. 2015;31(4):315-20. doi: 10.2116/analsci.31.315.
We report on the use of a peptide nucleic acid (PNA)-based fluorescent probe for the analysis of siRNA delivery to living cells. The probe, Py-AA-TO, possesses thiazole orange (TO) and pyrene moieties in the C- and N-termini of PNA, and can function as a light-up probe capable of selective binding to 3'-overhanging nucleotides of target siRNAs. The affinity-labeling of the siRNAs with Py-AA-TO facilitates fluorescence imaging of cellular uptake of polymer-based carriers encapsulating the siRNAs (polyplexes) through endocytosis and subsequent sequestration into lysosome. In addition, flow cytometric measurements reveal that the monitoring of Py-AA-TO fluorescence inside the cells is successfully applicable to the analysis of the polyplex disassembly. These promising functions of Py-AA-TO are presented and discussed as a basis for the design of molecular probes for fluorescent imaging and quantitative analysis of the siRNA delivery process.
我们报道了一种基于肽核酸（PNA）的荧光探针用于分析小干扰RNA（siRNA）向活细胞的递送。该探针Py-AA-TO在PNA的C端和N端分别具有噻唑橙（TO）和芘基团，可作为一种点亮型探针，能够选择性结合靶标siRNA的3'突出核苷酸。Py-AA-TO与siRNA的亲和标记有助于通过内吞作用对包裹siRNA的聚合物载体（多聚体）的细胞摄取进行荧光成像，并随后隔离到溶酶体中。此外，流式细胞术测量表明，监测细胞内Py-AA-TO荧光可成功应用于多聚体解离分析。Py-AA-TO的这些有前景的功能作为用于siRNA递送过程荧光成像和定量分析的分子探针设计基础进行了展示和讨论。
