title: FRET-based probing to gain direct information on siRNA sustainability in live cells: Asymmetric degradation of siRNA strands.
abstract: 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.
authors.affiliation: Department of Chemistry, Research Institute for Basic Sciences, Research Center for New Nano Bio Fusion Technology, Kyung Hee University, Seoul 130-701, Korea.
authors.forename: Seonmi
authors.initials: S
authors.lastname: Shin
authors.forename: Hyun-Mi
authors.initials: HM
authors.lastname: Kwon
authors.forename: Kyung-Sik
authors.initials: KS
authors.lastname: Yoon
authors.forename: Dong-Eun
authors.initials: DE
authors.lastname: Kim
authors.forename: Sang Soo
authors.initials: SS
authors.lastname: Hah
mesh_terms: D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D015336:Molecular Probe Techniques; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex