Live cell imaging of duplex siRNA intracellular trafficking

Intracellular distribution of siRNA after in vitro trans-fection 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 ob-servation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, fol-lowed, 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 specif-ically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point defini-tion of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear struc-tures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst re-lease into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Com-bination with a subsequent slower, but sustained re-lease from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture