In the field of DNA analyses, modified oligonucleotides containing fluorescent tags are widely used as molecular probes to detect single-nucleotide polymorphisms (SNPs). Detection is often based on FRET (fluorescence resonance energy transfer) as in Molecular Beacons. Another method relies on excimer emission (1, 2). Recently, we have applied exciplexes to the detection of mutations and SNPs. An emissive exciplex can be formed by the juxtaposition of two externally oriented exciplex-forming partners (X and Y) at the interface (nick region) of tandem oligonucleotides, which form a duplex on hybridization with their complementary target strand (Figure 1) (3). Addition of trifluoroethanol was required to facilitate exciplex emission, with exciplex emission optimal at ∼80% (v/v) of trifluoroethanol in the buffer. In the absence of trifluoroethanol for DNA-based probe oligos and targets there was no detectable exciplex emission, and few solvents could stand in for trifluoroethanol. A recent report (4) shows that if the exciplex partners are internally directed and lie within the body of the distorted DNA duplex, emission occurs even in the absence of added organic solvent. The present study is concerned with the origins of this difference in externally and internally oriented exciplex partners. Why is this specific trifluorethanol solvent medium so crucial in one family of exciplexes (here designated as externally directed), but not in the other, internally directed?



Figure 1: Diagram of the split-probe systems. For exciplexes, the A and B partners were based on pyrene and naphthalene respectively and for excimers both were pyrenyl.

Exciplex emission is generally strongly quenched as the solvent becomes more polar; for example, most organic exciplexes are non-emissive in media more polar than approximately acetontrile. In contrast, excimers are much less sensitive to their solvent polarity (5-8). In this contribution the very different response of excimers and exciplexes to increase in solvent polarity is used to study the effect of trifluoroethanol on such DNA-mounted systems and its control of exciplex/excimer emission. Hence, the sensitivity to trifluoroethanol composition of the pyrene-pyrene excimer and pyrene-dialkylnaphthalene exciplex of the systems in Figure 1 were studied and further probed using circular dichroism spectroscopy. Probes based on both DNA and LNA residues were used for the exciplex system, but only on DNA for the excimer. The results show that optimal fluorescence emission occurs in aqueous buffer containing approximately 80% v/v trifluoroethanol for both DNA- and LNA-based probe oligonucleotides forming exiplexes and also for excimers. This hints that the origin of the effect of the trifluoroethanol may not lie predominantly in the polarity effect on exciplex spectra. Circular dichroism spectroscopy data for these different systems supports an explanation that trifluoroethanol induces a structural transition to a different duplex structure, which is neither classical B- nor A-like, and once the favorable duplex has formed, the lowered polarity of this medium also favors exciplex emission.

References and Footnotes

1. Masuko, M., Ohtani, H., Ebata, K., and Shimadzu, A. Nucl Acids Res 26, 5409-5416 (1998).
2. Paris, P. L., Langenhan, J. M., and Kool, E. T. Nucl Acids Res 26, 3789-3793 (1998).
3. Bichenkova, E. V., Savage, H. E., Sardarian, A. R., and Douglas, K. T. Biochem Biophys Res Commun 332, 956-964 (2005).
4. Kashida, H., Komiyama, M., and Asanuma, H. Chem Lett 35, 934-935 (2006).
5. Birks, J. B. Photophysics of Aromatic Molecules. Wiley, London (1970).
6. Birks, J. B. Molecular Photophysics. Wiley , London (1973).
7. Rau, H. and Totter, F. J Photochem Photobiol A Chem 63, 337-347 (1992).
8. Knibbe, H. J Chem Phys 47, 1184-1185 (1967).