The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.
The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.
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荧光标记的 miRNA 通过疏水相互作用非特异性结合在细胞表面
DOI：
10.1371/journal.pone.0149751
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Zhang Q
中科院分区：
文献类型：
--
作者：
Lu T;Lin Z;Ren J;Yao P;Wang X;Wang Z;Zhang Q
背景MicroRNA是长约22 nt的小型非编码RNA，在几乎所有生物过程和疾病中发挥关键作用。荧光标记和脂质体转染是改变细胞内miRNAs水平和定位的两种常用方法。尽管人们对DNA/RNA脂质体转染的机制进行了大量的研究，但对荧光标记的miRNAs脂质体转染的特点、机制和特异性知之甚少。方法和结果因此，用不同的荧光染料标记的miRNA转染到贴壁和悬浮细胞使用脂质体试剂。采用流式细胞仪和激光共聚焦显微镜研究其非特异性结合及其机制。结果表明，Cy 5标记的miRNAs即使在不使用脂质体的情况下，也能与贴壁细胞（Hela）和悬浮细胞（K562）表面牢固结合。FAM标记的miRNAs与K562细胞结合明显，而与Hela细胞结合不明显。加入脂质体后，由于转染效率高，大部分与Hela细胞表面结合的荧光标记的miRNA被转染到细胞内，但大部分仍与K562细胞表面结合。结论荧光标记的miRNAs能够通过疏水作用与细胞表面发生非特异性结合，而高盐缓冲液对静电作用没有影响，有机溶剂对疏水作用有抑制作用。仅根据细胞荧光强度来估计转染效率会产生很大的误差。因此，应根据细胞类型采用其他方法评价转染效率，并选用更合适的荧光染料，以确保结果的准确性。
Background MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Methods and Results Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. Conclusions These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.