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

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 successfu...

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Bibliographic Details
Published in:Molecular bioSystems 2011-01, Vol.7 (7), p.2110-2113
Main Authors: Shin, Seonmi, Kwon, Hyun-Mi, Yoon, Kyung-Sik, Kim, Dong-Eun, Hah, Sang Soo
Format: Article
Language:English
Subjects:
Cell Survival
Confocal microscopy
Cytoplasm
Cytosol
DNA probes
fluorescence resonance energy transfer
Fluorescence Resonance Energy Transfer - methods
Fluorescent Dyes - metabolism
HEK293 Cells
Humans
Kinetics
Microscopy, Confocal
Molecular Probe Techniques
Oligonucleotides
RNA Stability
RNA, Small Interfering - metabolism
RNA-Induced Silencing Complex - metabolism
RNA-mediated interference
siRNA
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Summary: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.
ISSN:1742-206X
1742-2051
DOI:10.1039/c1mb05054k