In situ analysis of single-stranded and duplex siRNA integrity in living cells
In situ analysis of single-stranded and duplex siRNA integrity in living cells
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DOI：
10.1021/bi060351b
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发表时间：
2006-09-05
期刊：
影响因子：
2.9
通讯作者：
De Smedt, Stefaan C.
中科院分区：
文献类型：
--
作者：
Raemdonck, Koen;Remaut, Katrien;De Smedt, Stefaan C.
为了实现短干扰RNA（siRNA）的全部治疗前景，认为诸如增加的生物稳定性的改进是关键的。遗憾的是，迄今为止，关于siRNA的细胞内稳定性的原位数据不足。我们报告使用先进的荧光为基础的方法来探测双标记的siRNA，这是受荧光共振能量转移（FRET）的溶核衰变。用RNA酶A和细胞提取物进行的体外测量表明，受体（5 '-Cy 5）与供体（3'-罗丹明绿色）荧光的比率可用于研究供体激发后标记的siRNA底物的降解。细胞内FRET分析显示单链siRNA的大量降解，而双链体siRNA在测量的时间段内保持完整。这些数据强调了未修饰的双链体siRNA的高内在核酸酶抗性，并证明细胞持久性对于单链结构更为关键。首次在活细胞内实时研究siRNA的稳定性。这里提出的基于荧光的方法是一种直接获得活细胞内siRNA完整性信息的技术，并为了解更多关于siRNA治疗剂的细胞内命运提供了光明的前景。
To attain the full therapeutic promise of short interfering RNA ( siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer ( FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.