The creation of reliable and highly specific methods for in situ detection of nucleic acids and other biomacromolecules is crucial for development of novel approaches in genomic and post-genomic molecular diagnostic (1-3). Recently we reported the evidence of self-assembly of heterobinuclear d-f complexes in protic media, using d-block complexes as potential sensitizers, and lanthanide ions acting as potential energy acceptors (4). This approach provided a basis for potential application of visible metal-to-ligand charge-transfer (MLCT) luminescence to DNA probes, sensors and bio-assays. We report here the first example of the use of a pair of metallo-chromophores covalently attached to oligonucleotide split-probes to detect mutations and mismatches in short DNA fragments via resonance energy-transfer on target-directed assembly. Octahedral tris-chelate complexes [M^II(bpy)₃]²⁺ (M = Ru or Os, bpy = 2,2'-bipyridyl), covalently attached to the 3'- and 5'-phosphates of two oligonucleotides, are juxtaposed when hybridized contiguously to a fully complementary DNA target. Visible metal-to-ligand charge-transfer excitation of the [Ru^II(bpy)₃]²⁺ unit leads to resonance energy-transfer (ET) to the MLCT state of the [Os^II(bpy)₃]²⁺ moiety. The extent of attenuation of the intense red luminescence from the Ru^II chromophore allows highly sensitive structural probing of the assembly and constitutes a novel approach to DNA detection and mismatch discrimination.


Figure 1: Target-directed self-assembly of the split-probe DNA system functionalized by metallochromophore parners. D and A represent the [Os^II(bpy)₃]²⁺ and [Ru^II(bpy)₃]²⁺ partners attached through linkers to respective oligonucleotides via the 5' or 3' terminal phosphate groups.

References and Footnotes

1. Wang, J. Nucleic Acids Res. 28, 3011?3016 (2000).
2. Palecek, E. and Fojta, M. Anal. Chem. 73, 75A?83A (2001).
3. Drummond, T. G., Hill, M. G., and Barton, J. K. Nat. Biotechnol. 21, 1192?1199 (2003).
4. Pope, S. J. A., Coe, B. J., Faulkner, S., Bichenkova, E. V., Yu, X., and Douglas K. T. J. Am. Chem. Soc. 126, 9490?9491 (2004).