Live cell imaging of duplex siRNA intracellular trafficking

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.

Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene

“…Application of different siRNA complexes with lipids and polymers faces with the need to address different requirements for effective formation of the complex, its stability in serum, and effective release from the endosome, which necessitates construction of multilevel structures 40 . The issue of toxicity is also very important 41 .…”

“…In all cases, siRNA within the cell is isolated from the cytoplasm by the endosomal membrane 40 . Endosomal escape is a limiting step in achieving effective gene silencing 59 .…”

Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex

“…A fraction of the siRNA translocate into the nucleus, which is in accordance with a previous report. 31 A very small fraction of the siRNA remains complexed with streptavidin and entrapped in lysosomes. A significant amount of siRNA and nanocomplex are exocytosed, which may explain the transient silencing activity of synthetic siRNA delivered by nonviral vectors.…”

“…37, 38 In accordance with our results, several other groups also demonstrated that siRNA alone can translocate inside and out of the nucleus. 31, 39 …”

How can Novel Microscopic Approaches Shed Light on the Function of Nucleic Acid-Based Drugs?

“…Editorial kinetic parameters associated with siRNA diffusion, dissociation and degradation [17]. Using FRET-labeled siRNA as a method for assaying siRNA integrity, it was shown that the cytoplasmic delivery of siRNA is accomplished by burst release from endosomal vesicles [18].…”