Cellular distribution of intact siRNA

RNAi is a powerful tool for analyzing gene function and promises high potential for therapeutic application. While in cell culture models efficient knockdown rates can be achieved, applications in vivo remain challenging. Especially the delivery of siRNA molecules inside the target cell and a progressing degradation of siRNA applied to the target organism or cell, are the major hurdle for therapeutic application. We have already shown that double labelled siRNA can be used for integrity measurements of duplex siRNA in the cuvette and in fixed cells [1]. These measurements are based on the ratio of red and green emission of a FRET-dye-pair: Fluorescein or Atto488 as FRET-donor and Tetramethylrhodamine or Atto590 as FRET-acceptor. The FRET-dyes are attached on the sense and on the antisense strand of an siRNA duplex and the proximity of both strands can then be monitored by measuring the occurring FRET [2, 3]. The system is sensitive enough to detect degradation levels of as little as 5 % of the labelled siRNA. In fixed cells a reservoir of intact siRNA could be detected up to 10 days after transfection, showing a shift in cellular distribution from the cytoplasm into nucleus and back to the cytoplasm. With Atto488 and Atto590 we identified a photostable FRET-dye-pair alternative that can also be used for long-term imaging in living cells, enabling us to monitor the uptake of labelled siRNA into the cell and a subsequent release from the transfection particles. The live-cell imaging indicates that the release of transfected siRNA is a random process and changes in cellular distribution can be observed within few minutes.