FRET-based probing to gain direct information on siRNA sustainability in live cells: Asymmetric degradation of siRNA strands

Department of Chemistry, Research Institute for Basic Sciences, Research Center for New Nano Bio Fusion Technology, Kyung Hee University, Seoul 130-701, Korea.
Molecular BioSystems (Impact Factor: 3.21). 01/2011; 7(7):2110-3. DOI: 10.1039/c1mb05054k
Source: PubMed


Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.

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    • "Numerous reports illustrate that application of FRET to siRNA has a high potential to elucidate details of siRNA traffi cking (Raemdonck et al. , 2006 ; Jarve et al. , 2007 ; Jiang and Zhang , 2010 ; Kim et al. , 2010 ; Lee et al. , 2010 ; Hoerter et al. , 2011 ; Shin et al. , 2011 ). Apart from intrasiRNA FRET, energy transfer between nanocarriers and the siRNA is another elegant means that seems particularly suited to unravel details of release and unloading of the siRNA payload inside the cells (Jiang and Zhang , 2010 ; Lee et al. , 2010 ; Raemdonck et al. , 2010 ). "
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