(Received for publication, August 16, 1995)
From the
A method was investigated for monitoring the integrity of oligonucleotides in solution and in cells using fluorescence resonance energy transfer between two different fluorochromes attached to a single oligonucleotide. Ten-mer oligodeoxyribonucleotides labeled with fluorescein at one end and with rhodamine X at the other end were used. The oligomer had a specific absorption spectrum with peaks at 497 and 586 nm, which corresponded to fluorescein and rhodamine X, respectively. When excited at 494 nm, the oligomer had a specific fluorescence spectrum with peaks at 523 and 610 nm. The fluorescence intensity at 610 nm was 6-8 times higher than that at 523 nm. After digestion of the oligomer with an endonuclease, the fluorescence at 523 nm increased more than 12-15-fold but its fluorescence peak at 610 nm almost completely disappeared. To examine effects in vivo, sea urchin eggs were injected with a solution of the oligomer 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. The ratio of the intensities of red to green fluorescence decreased in dependence on time after injection of the oligomer. These changes were not observed in eggs that had been injected with a solution of similarly double-labeled, phosphorothioate oligomer. These results indicated that unfertilized sea urchin eggs had nucleolytic activity. Analysis in vitro on supernatant of the egg homogenate indeed demonstrated the existence of nucleases. All together, our results indicate that the integrity of oligonucleotides can be estimated in living cells by monitoring the fluorescence resonance energy transfer of the double-labeled oligonucleotide.
The injection of oligonucleotides into living cells is an
effective method for control of the expression of target
genes(1, 2) . Oligodeoxyribonucleotides have been used
to analyze the functions of various genes, since such short DNA
molecules can inhibit the synthesis of particular proteins in injected
cells by binding to the corresponding mRNA that includes the
complementary sequence(3, 4) . Introduction of a
functional gene with transposable elements into target cells would
allow us to produce functionally important cell lines(5) . The
fate of single-stranded oligonucleotides in vivo has been
monitored by use of materials labeled with a fluorescent dye at one
end(6) . In the search for a treatment for AIDS, ribozymes or
their artificially modified analogues appear to be good candidates for
drugs of the future since specially designed ribozymes can specifically
cleave the mRNA of the AIDS
virus(7, 8, 9, 10, 11, 12, 13) .
In studies of such possible therapeutic modalities, the structural
integrity of oligonucleotides merits careful consideration because of
the dependence of specific binding activity on the length of the
oligonucleotide sequence. There is always the undesirable possibility
of depolymerization or cleavage by digestion by intracellular
nucleases. Ribozymes are especially sensitive to intracellular
ribonucleases because their active sites are composed of
RNA(14) . If we are to monitor the fate of ribozyme in vivo by use of conventional materials labeled with a fluorescent dye at
one end, we are likely to detect degraded RNAs labeled with a
fluorescent dye at their end. To address these issues, we investigated
methods for estimating the integrity of injected oligonucleotides and
detecting the nucleolytic activity in living cells using
oligonucleotides that had been double-labeled with two fluorescent
dyes. We examined the fluorescence characteristics of and the
fluorescence resonance energy transfer (FRET) ()within these
double-labeled oligonucleotides.
FRET is an interesting example of a fluorescence-related phenomenon (15, 16) . When the fluorescence spectrum of one fluorochromes, the donor, overlaps with the excitation spectrum of another fluorochrome, the acceptor, and when the donor and the acceptor are in close physical proximity, the excitation of the donor induces the emission of fluorescence from the acceptor as if the acceptor has been excited directly and the intensity of fluorescence from the donor decreases. The extent of FRET is extremely sensitive to the distance between the donor and the acceptor, being inversely proportional to the sixth power of the distance. This phenomenon can be explored for studies of intermolecular and intramolecular relationships in biophysical investigations and in cell biology and has been used, for example, to examine the structure in solution of a hammerhead ribozyme(17) , the concentration of cAMP in vivo(18) , membrane fusion(19) , retroviral proteases(20) , nucleic acid structures and sequences(21) , and the extent of intracellular oligonucleotide hybridization(22) , kinetic studies of hybridization to the oligonucleotide(23) , donor-acceptor distance distributions in a double-labeled fluorescent oligonucleotide(24) .
To investigate the integrity of oligonucleotides and the detection of nucleolytic activity in living cells, we used fluorescently double-labeled, single-stranded oligodeoxyribonucleotides. FRET was demonstrated spectroscopically in solutions of the modified oligonucleotides and was visualized under the fluorescence microscope in sea urchin eggs after microinjection of the modified oligonucleotides. Enzymatic digestion eliminated the efficiency of FRET both in solutions and in eggs. These results indicate that it is possible to detect only the intact oligonucleotides in living cells by monitoring FRET. We also found nucleolytic activity in living, unfertilized sea urchin eggs by measuring the time-dependent decrease of efficiency of the FRET.
Alternatively, we also used a chemically synthesized 10-mer oligodeoxyribonucleotide (5`-TGAAATTGTT-3`) that was double-labeled with fluorescein at its 5`-end and rhodamine X at its 3`-end, respectively (F-ODN-R) and we also used another chemically synthesized 10-mer phosphorothioate oligodeoxyribonucleotide (5`-TGAAATTGTT-3`) that was double-labeled with rhodamine X at its 5`-end and fluorescein at its 3`-end, respectively (R-S-ODN-F). F-ODN-R was supplied by Takara Shuzo (Kyoto, Japan) and R-S-ODN-F was supplied by Bex (Tokyo, Japan). The purity of F-ODN-R was confirmed by HPLC under the conditions described above. These fluorescent oligomers were confirmed to have the same spectrometric characteristics as the enzymatically synthesized oligomer.
Figure 1: Chromatogram of a solution of F-ODN-R. Chromatography was performed on an ion-exchange column with a 30-min linear gradient from 100 to 1,000 mM NaCl in Tris-HCl (pH 9.0) at a flow rate of 1.0 ml/min. This chromatogram indicated that the preparation of F-ODN-R was composed of a single oligonucleotide.
Figure 2: Absorption spectrum of F-ODN-R in medium A. The peaks at 497 and 586 nm indicate that the solution included fluorescein and rhodamine X.
Figure 3: Fluorescence spectra of F-ODN-R in medium A. A, emission spectrum of a solution of F-ODN-R, upon excitation at 494 nm (band-pass, 5 nm). This spectrum has peaks at 523 and 610 nm. The peak at 523 nm corresponds to the fluorescence of fluorescein, and the peak at 610 nm corresponds to that of rhodamine X. B, excitation spectrum of a solution of F-ODN-R, monitored at 606 nm (band-pass 5 nm). This spectrum has peaks at 500 and 594 nm. The peak at 500 nm corresponds to the absorbance of fluorescein, and the peak at 594 nm corresponds to that of rhodamine X. The fluorescence spectra of F-ODN-R in medium A indicate that FRET occurred in the solution of F-ODN-R.
To confirm that the observed FRET occurred between the dyes attached to one and the same molecule of the oligonucleotide, we digested the oligonucleotide with Bal-31, which functions as an endonuclease against single-stranded DNA. After incubation with the enzyme, the digested material was analyzed spectroscopically. The fluorescence intensity at 523 nm was 15 times higher than that before endonucleolytic digestion (Fig. 4). The peak of red fluorescence at 610 nm disappeared. These results indicate that FRET of the intact oligomer could be ascribed to the relationship between intramolecular fluorochromes. Therefore, it appeared that the integrity of the oligonucleotide could be recognized from the changes in the fluorescence intensity of the donor and the acceptor fluorochromes, and that it should be possible to infer, from in vivo measurements of FRET, the degree of integrity of double-labeled fluorescent oligonucleotides.
Figure 4: Fluorescence spectrum of products of the reaction with endonuclease (Bal-31) in medium A (solid line). The digested F-ODN-R had a peak of fluorescence at 523 nm that corresponded to fluorescein. The intensity of fluorescence was 15 times higher than that before endonucleolytic treatment. The peak of red fluorescence at 610 nm corresponding to rhodamine X has disappeared. These results indicate that the FRET of F-ODN-R was due to the relationship between intramolecular fluorochromes. Spectrum indicated by the dotted line is a spectrum of a solution of F-ODN-R before treatment with Bal-31 (same spectrum as shown in Fig. 3A).
Figure 5: Fluorescence microscopy of the sea urchin eggs that had been microinjected with the double-labeled fluorescent oligonucleotide F-ODN-R. Unfertilized eggs were injected with the intact oligomer (A, B, and F) or with the enzymatically digested oligomer (D and E). The paired images of fluorescent components in the green-light region (A and D) and the red region (B and E) were recorded separately from the same eggs. The ratio image (C) of red to green was computed from the fluorescent components in photographs A and B. These photographs are shown in pseudo colors. Photograph F shows the transmitted-light image of the egg. Bar equals 50 µm.
The intensity of fluorescence varied among different regions of the egg. The middle region of the egg was intensely fluorescent and the intensity of fluorescence decreased from the center to the periphery in both red- and green-light images. The absence of uniformity can be ascribed to the length of the light path within the spherical sea urchin egg. The ratio intensities of the two qualitatively different images could be used to eliminate the effect of cell volume, as in the case of calculations of intracellular concentrations of calcium ions using the fluorescent dyes Fura-2 (31) and Indo-1(32) . We ``divided'' the red image by the green image with an image processor and the result is shown in Fig. 5C. The new image shows the uniformity of FRET within the cytoplasm of an egg injected with F-ODN-R. The cytoplasmic margin of the egg surface, namely, the egg cortex, gave a higher value for the ratio than the inner cytoplasm. This higher ratio might be the result of the higher viscosity in the narrow cortex region as compared with the inner region(33) . Such a difference causes the disparity in the refraction between green and red light at the surface, giving rise to an error in the estimation of FRET. While we were unable to clarify the discontinuity in the extent of FRET at the cortex, the ratio of the images demonstrates that the double-labeled fluorescent oligonucleotide exhibited uniform FRET throughout the cytoplasm of the living egg.
The
elimination of FRET after cleavage of the oligonucleotide was
demonstrated by injection of F-ODN-R that had been pretreated with
endonuclease (Fig. 5, D and E). If the sea
urchin egg contains nucleases, the injected double-labeled oligomer
should be degraded and the extent of FRET should decrease with the time
after the injection. Therefore, we measured time-dependent changes in
the fluorescence ratio of the red to the green component from a single
living egg under the microscope (Fig. 6A). When the
oligonucleotide was injected, FRET rapidly decreased and reached
plateau in about 1 h. The ratio decreased from 2.3 at 1 min to 0.42 at
60 min. This time-dependent decrease of FRET was not observed in the
eggs injected with double-labeled phosphorothioate, R-S-ODN-F, which is
known to be resistant to the digestion by general nucleases. This
modified R-S-ODN-F had the same FRET property as that of the oligomer
with regular phosphodiester linkages (Table 1): note that the
fluorescence ratio of R-S-ODN-F decreased by the treatment with
nuclease P1, a nucleolytic enzyme that can digest phosphorothioate
linkages with S configuration(29) . The
incubation of the eggs injected with phosphorothioate R-S-ODN-F for 60
min showed no change in the ratio (Fig. 6B). These
results indicate that the lower extent of FRET in the eggs for the
double-labeled oligomer with natural phosphodiester linkages was mainly
due to the enzymatic digestion of the oligonucleotide.
Figure 6: Time course of ratio of the red to the green components of the fluorescence in eggs under the fluorescence microscope. The ratio of the two-color components in eggs injected with R-ODN-F, that has natural phosphodiester linkages, gradually decreased in 60 min (A). The ratio of the two-color components in eggs injected with R-S-ODN-F, that has nuclease-resistant phosphorothioate linkages, was constant over 60 min (B). Fluorescence measurements under microscope started at 1 min after injection of double-labeled oligonucleotides.
Figure 7: Chromatogram for detection of nucleolytic activity in the supernatant of unfertilized sea urchin egg homogenate. Digestive products from 9-mer oligonucleotide conjugated with rhodamine X at its 5`-end (R-ODN) were detected by fluorescence of rhodamine X, monitored at 610 nm upon excitation at 585 nm. Eight peaks were detected in the digestive reaction mixture (A). A single peak was detected in the same reaction mixture, in that digestive reaction was inhibited by an addition of EDTA (B). Intact R-ODN showed the identical chromatogram to B. These results indicate that R-ODN was digested by egg supernatant and that nucleolytic activity was inhibited by EDTA.