Detection of undegraded oligonucleotides in living sea urchin eggs by fluorescence resonance energy transfer.

A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. A 10-meric oligodeoxynucleotide labeled with fluorescein at its 5'-end and with rhodamine X at its 3'-end (F-ODN-R) was used. The oligomer had a specific absorption spectrum with peaks at 497 nm and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, F-ODN-R had a specific fluorescence spectrum with peaks at 523 nm and 610 nm. The digestion of F-ODN-R with an endonuclease caused the increase in light intensity at 523 nm and the decrease at 610 nm. To examine effects in vivo, living sea urchin eggs were injected with a solution of F-ODN-R and excited with blue light at 470-490 nm. Two fluorescent images, a green image at 520-560 nm and a red image at above 580 nm, were obtained when a single egg was viewed under a fluorescence microscope. Eggs injected with the digested F-ODN-R emitted only green fluorescence. These results indicated that the integrity of oligonucleotides can be estimated in living cells by monitoring FRET after double-labeling of the oligonucleotides with fluorescein and rhodamine X.