Development of a Time-Resolved Fluorometric Method for Observing Hybridization in Living Cells Using Fluorescence Resonance Energy Transfer

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.

Rare earth cryptates for the investigation of molecular interactions in vitro and in living cells

“…The use of time-resolved fluorescence microscopy to detect FRET was previously shown to be feasible using a microinjection with oligonucleotides labeled with Bodipy/Cy5 pair [13], this choice of donor imposed the use of a picosecond fluorescent system and Ti-Sapphire laser. The use of FRET donor such as the lanthanide cryptate described herein allow us to work with a longer decay (100 s range) and therefore less costly equipment.…”

mRNA detection in living cell using phosphorothioate-modified molecular beacon

“…As shown in Figure 4, the changing of fluorescence intensity can preferably reflect the concentration difference of S-cDNA. The concentration and microinjection volume of the preformed duplex were 5 μmol/L (S-GFP-MB) and 0.015 pL (about 1% volume of the cell) [37] , respectively. The limit of detection was 3.33 nmol/L equivalent to 10 3 copies per cell.…”

Advances in Fluorescent Tracking of Nucleic Acids in Living Cell

“…Tsuji and colleagues (21) were among the first to test this FRET approach for imaging nucleic acids in living cells by visualizing the expression of c-fos mRNA in transfected COS-7 cells. A little later, they visualized the presence of c-fos mRNA in HeLa cells by FRET, measuring acceptor fluorescence decays with a time-resolved fluorescence microscope (22). Disappointingly, however, the application of FRET did not remove the limitations associated with the use of ODNs in in vivo hybridization studies.…”