ArticleBiophysical journal2001
Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.
Article in Biophysical journal, 2001. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 57 citations in OpenAlex.
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- Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules.eLife · 2019Article
- A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA editChemical science · 2018Article
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- Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.Organic & biomolecular chemistry · 2013Article
- Molecular beacons of xeno-nucleic acid for detecting nucleic acid.Theranostics · 2013Review
- Comprehensive study in the inhibitory effect of berberine on gene transcription, including TATA box.PloS one · 2011Article
- A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization.Nucleic acids research · 2009Article
- Fluorescent probes for live-cell RNA detection.Annual review of biomedical engineering · 2009Review
- Synthesis of peptide nucleic acid FRET probes via an orthogonally protected building block for post-synthetic labeling of peptide nucleic acids at the 5-position of uracil.Bioconjugate chemistry · 2008Article
- Two-photon excited fluorescence energy transfer: a study based on oligonucleotide rulers.Journal of fluorescence · 2006Article
- Imaging of single mRNAs in the cytoplasm of living cells.Progress in molecular and subcellular biology · 2004Review
- Structure-function relationships of shared-stem and conventional molecular beacons.Nucleic acids research · 2002Article
- The dynamic pathway of nuclear RNA in eukaryotes.Nucleus (Austin, Tex.)Review
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.
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