RNAi promises high potential for therapeutic application by enabling temporary specific gene knockdown. However applications in vivo remain challenging, with delivery and progressing degradation of siRNA being the major hurdle.
We use siRNA labeled with two fluorescent dyes to analyze the integrity of the duplex and furthermore the cellular uptake and distribution. Integrity measurements are based on FRET occurring between a Donor dye (Atto488) and an acceptor dye (Atto590), which are attached to sense and antisense strand Donor
of the siRNA duplex. With degradation, the FRET signal collapses and the ratio of FRET- to donor-signal (R/G-ratio) decreases.
The R/G-ratio can be visualized in confocal imaging data to analyze integrity inside cells and thus get insight in the dynamics of siRNA inside a cell.