title: Poly(A)-targeting molecular beacons: fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells.
abstract: Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)(+)-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25nM to 0.5μM) of poly(A)-RNA, as well as for determination of K(d) values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed.
authors: Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea.
authors: Min Young
authors: MY
authors: Kim
authors: Jisu
authors: J
authors: Kim
authors: Sang Soo
authors: SS
authors: Hah
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D011061:Poly A / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D013997:Time Factors
chemical_list: D002232:Carbocyanines; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; C089187:cyanine dye 3; C085321:cyanine dye 5; D011061:Poly A