Abstract
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.
Highlights
SiRNA-based RNAi has become a powerful tool for studying gene function in vitro and promises to be a versatile tool for in vivo application and therapy [1]
Synthetic siRNA is delivered by delivery agents of cationic origin typically via endocytosis or related pathways [5,6,7,8] and results in the formation of an active RNA induced silencing complex (RISC) that consists of an Argonaute protein as major effector, one strand of the former siRNA duplex, and helper proteins, like TRBP and Dicer [9,10,11]
As a motivation for this study, we argue that the kinetics of siRNA uptake and release in the cell are ill understood, and that their investigation using widespread transfection agents is likely to shed light on parameters that contribute to the success of these agents
Summary
SiRNA-based RNAi has become a powerful tool for studying gene function in vitro and promises to be a versatile tool for in vivo application and therapy [1]. Work relied on the classical fluorescein/tetramethylrhodamine (FL/TMR) FRET pair, while more recently developed dyes and dye combinations offer improved spectral properties In this respect, our recent screening of various FRET pairs identified Atto 488/Atto 590 labeled siRNA duplexes, with Atto 488 as donor dye on the 3 -end of the passenger strand and Atto 590 as acceptor dye on the 5 -end of the guide strand (Figure 1A) as optimized combination with respect to spectral parameters including spectral separation, FRET efficiency, bleed through, cross excitation and photobleaching [35]. This suggests that, at least in cell culture, the molecular basis for efficient RNAi relies on intracellular distribution events taking place during the initial 4 h after in vitro transfection
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