Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
Biol Chem. 2012 Jan;393(1-2):23-35. doi: 10.1515/BC-2011-256.
Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.
研究小干扰 RNA（siRNA）转染后的命运可能揭示提高 RNA 干扰（RNAi）效率的新方法。由于 RNA 的细胞内降解可能阻止对荧光标记 siRNA 的可靠观察，因此需要新的荧光显微镜工具来覆盖 RNAi 效应的相当长时间。本文报道了用于感测双链 siRNA 完整性的新型荧光共振能量转移（FRET）染料对的特性和应用，通过活细胞成像可以评估 siRNA 细胞群体的降解状态。研究了一组高产荧光染料，以评估它们作为用于研究细胞内 RNA 的 FRET 对的适用性。基于光稳定性、交叉激发、串扰以及由于 RNA 链杂交和 pH 变化等引起的荧光定量变化等测定中的性能，评估了 13 对 FRET 对中的 9 个染料。已经将 Atto488/Atto590 FRET 对应用于活细胞成像，并首次揭示了完整 siRNA 异常转运的一些方面。时程研究显示 siRNA 在大核周结构中高度动态运动。这些和由此产生的优化的 FRET 标记 siRNA 有望对未来在活细胞中观察标记 RNA 产生重大影响。
