Dye selection for live cell imaging of intact siRNA.

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) effi ciency. Because intracellular degradation of RNA may prevent reliable observation of fl cence-labeled siRNA, new tools for fl uorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fl uorescence 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 fl uorescent 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, bleedthrough, as well as on quantifi ed changes of fl 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 fi rst aspects of unusual traffi cking 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 signifi cant impact on future observations of labeled RNAs in living cells.