Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer

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 ol igodeoxynucleotides, each labeled with donor or acceptor, into the cytoplas m, making them hybridize to adjacent locations on c-fos mRNA, and taking im ages of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimo to, Y. Sate, M. Hirano. Y. Sei-lida, S. Kondo, and K. Ishibashi, 2000, Biop hys. 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 i n living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hyb ridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. Wh en a combination of a donor having longer fluorescence lifetime and an acce ptor having shorter lifetime is used, the measured fluorescence decays of a ccepters under FRET becomes slower than the acceptor fluorescence decay wit h direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the t arget RNA has no single-strand part between the two fluorophores, the accep tor fluorescence of hybrid had a sufficiently longer delay to detect fluore scence of hybrid in the presence of excess amounts of non-hybridizing probe s. 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 cel l autofluorescence in the cytoplasm of living cells under a time-resolved f luorescence microscope with a time-gated function equipped camera. The pres ent method will be useful when observing induced expressions of mRNA in liv ing cells.