Hirsch Markus, Helm Mark
Institute of Pharmacy and Biochemistry, University of Mainz, D-55128 Mainz, Germany Institute of Pharmacy und Molecular Biotechnology, University of Heidelberg, D-69120 Heidelberg, Germany.
Institute of Pharmacy and Biochemistry, University of Mainz, D-55128 Mainz, Germany Institute of Pharmacy und Molecular Biotechnology, University of Heidelberg, D-69120 Heidelberg, Germany
Nucleic Acids Res. 2015 May 19;43(9):4650-60. doi: 10.1093/nar/gkv307. Epub 2015 Apr 13.
Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.
体外转染后，小干扰RNA（siRNA）的细胞内分布通常取决于脂质多聚体，脂质多聚体必须将siRNA释放到细胞质中。在此，通过基于荧光共振能量转移（FRET）的活细胞成像监测了siRNA的命运。在对摄取和释放过程进行原位观察之后，这种方法能够观察到许多迄今为止尚未表征的细胞内分布和降解过程，首先是一阵内体释放，在某些情况下，随后是siRNA快速流入细胞核。通过专门开发的成像算法，可以在由高级降解产生的游离荧光团背景下持续观察完整的siRNA，该算法基于FRET在像素中识别完整siRNA群体。这在siRNA分布的终点定义中被证明是至关重要的，siRNA分布的典型特征是在核周结构中存在部分降解的siRNA池。我们的结果表明最初的4小时是一个关键的时间窗口，其特征是快速初始爆发性释放到细胞质中，这为随后siRNA的细胞内分布奠定了基础。与随后从内体储存库中较慢但持续的释放相结合，可能有助于RNA干扰（RNAi）的效率和持续时间，并解释了脂质多聚体在细胞培养RNAi实验中的成功。
