For large genomes (10⁶-10⁹ bp, depending on the species) oligonucleotide probes presently used are at least 17 to 20 nucleotides residues long, and often longer, to avoid random, non-specific base pairing of regions of the target with probe. Commonly, to minimize background fluorescence the components of the final emissive fluorophore are in two pieces, relying on Fluorescence Resonance Energy Transfer (FRET). The background can be further decreased using split oligonucleotide systems that deliver components of a final fluorophore that are separately non-fluorescent at the detection wavelength, e.g., two short oligonucleotides hybridizing contiguously at their target sequence, bringing two pyrene groups together to form a pyrene excimer (Figure 1; A = B = pyrene), which emits with a very large Stokes shift (∼130 nm) (1, 2). The scope for chemical variation using only excimers is limited and we introduced non-identical partners A and B for such nucleic acid systems, i.e., exciplexes (3). Conditions leading to exciplex emission for the DNA system were discovered by use of buffers that contained 80% (v/v) trifluoroethanol.



Figure 1:Diagram of the split-probe excimer (A=B) and exciplex (A≠B) systems.

We now describe the effects of incorporation of locked nucleic acid (LNA) residues in the probe oligonucleotides in exciplex-based fluorescence detection of DNA. The system studied consisted of two 8-mer oligonucleotides, complementary to adjacent sites of a 16-mer DNA target. Each probe oligonucleotide contained various combinations of LNA residues (0, 2, 3, or 8), tested for their efficacy in forming an emissive exciplex on correct hybridisation to the DNA target. The exciplexes formed emitted at ∼480 nm with excited at 350 nm with large Stokes shifts (∼130-172 nm). The best combination of LNA and DNA residues in the probes was two LNA residues in the 3?-probe and three in the 5?-probe.

References and Footnotes

1. Paris, P. L., Langenhan, J. M., and Kool, E. T. Nucleic Acids Research 26, 3789-3793 (1998).
2. Ebata, K., Masuko, M., Ohtani, H., and Kashiwasake-Jibu, M. Photochem Photobiol 62, 836-839 (1995).
3. Bichenkova, E. V., Savage, H. E., Sardarian, A. R., and Douglas, K. T. Biochemical and Biophysical Research Communications 332, 956-964 (2005).