Real-time analysis of siRNA integrity and distribution

RNAi is a powerful tool for analyzing gene function and promises high potential for therapeutic application. Whereas efficient knockdown rates can be achieved in cell culture, applications in vivo remain challenging. Especially the delivery of siRNA molecules into the target cell and a progressing degradation of siRNA applied to the target organism or cell, are the major hurdles for therapeutic application. Double labeled siRNA has been used as a classifier for siRNA integrity, both in the cuvette and in fixed cells [1]. FRET, occurring between two fluorescent dyes attached to the ends of both strands of an siRNA duplex, serve as a sensitive indicator for strand separation and the degradation of siRNA. The integrity measurements are based on the ratio of red and green emission of a FRET-dye-pair (e.g. Atto488 / Atto590 or Fluorescein / Tetramethylrhodamine). The system is very sensitive and enables the detection of degradation levels of as little as 5 % of the labeled siRNA [2, 3]. With Atto488 and Atto590 a photostable FRET-pair was established that allows long-term observation inside living cells and thus offering the possibility to monitor the uptake, release and dynamics of labeled siRNA. Live-cell imaging indicates that the release of transfected siRNA is a random process and changes in cellular distribution can be observed within a few minutes. Further studies indicated a long survival of duplex siRNA inside cells, which may serve as a reservoir for prolonged RNAi.