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.

Abstract— We developed a novel nucleic acid hybridization method based on excimer formation. We used two different 16‐mer oligonucleotide probes that had a combined continuous‐sequence run that was complementary to a target 32‐mer. Prior to hybridization, the adjacent terminal ends (i.e. the 3'‐terminal of one probe and the 5'‐terminal of the other probe) were each labeled with one pyrene residue. When these probes simultaneously hybridized to the target, a 495 nm broad fluorescence band was produced. The intensity of this band increased as the intensity of the pyrene monomer bands decreased, indicating that the 495 nm band was attributed to the pyrene excimer. The excimer fluorescence, easily differentiated from the monomer bands for emission wavelength, opens up a new way to perform homogeneous hybridization assays and in vivo imaging of nucleic acids.

The authors have developed a noninvasive optical method to image the subarachnoid space and cerebrospinal fluid pathways in vivo based on the near-infrared fluorescence of indocyanine green (ICG). The ICG was bound to purified lipoproteins (ICG-lipoprotein) and injected into the subarachnoid space of neonatal and adult rats. The ICG fluorescence was detected by a cooled charge-coupled device camera. After injection of ICG-lipoprotein into the cerebral subarachnoid space of the neonatal rat, ICG fluorescence was clearly detected at the injection site through the skull and skin. The ICG fluorescence was observed in the cerebellum and the lumbar spinal cord 1 and 8 hours postinjection, respectively. After injection of ICG-lipoprotein into the lumbar spinal subarachnoid space of an adult rat, ICG fluorescence was observed from the injection site to the thoracic levels along the spinal subarachnoid space. In addition, with the rat's head tilted downward, ICG fluorescence had extended to the cerebral subarachnoid space by 1 hour postinjection. The ICG fluorescence imaging of the cerebral subarachnoid space demonstrated an increase in fluorescence intensity around the lambdoid suture and the forebrain. On dissection of the rat brain the former location was identified as the supracerebellar cistern and the latter as the olfactory cistern. The results of this study are the first to demonstrate that an optical technique is applicable to imaging of the subarachnoid space and cerebrospinal fluid pathways in vivo. In addition, ICG-lipoprotein provides a sensitive optical tracer for imaging extravascular biological structures. Finally, ICG fluorescence imaging does not require an intricate imaging system because ICG is localized near the surface of the body.

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