Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.

The tetradeoxyribonucleotide pAGCG in 1 M NaCl forms duplexes with terminal non-canonical pAWG pairs with stability significantly exceeding that for the duplex (pAGCT) P and lower than that for the duplex (pCGCG) P . The deoxyriboduplex (pAGCG) P is considerably stabilized by 3P-Y and slightly by 5P-X dangling bases. Therefore, the stability of duplexes with 3P dangling bases decreases in the order (pAGCGY) P s (pCGCGA) P s (pAGCTA) P . The sum of the independent stabilizing effect of the of 5P-pG and 3P-A dangling bases on the (pAGCG) P core duplex is higher than that of the additional terminal pGWA pair in pG-A-/-G-A tandem of the duplex (pGAGCGA) P .z 1997 Federation of European Biochemical Societies.

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New pyranone derivatives having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5'-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleotide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response. The Tm data and thermodynamic parameters for complex formation have demonstrated the ability of chromone (gamma-pyrone) and coumarin (alpha-pyrone) derivatives to stabilize strongly 7-mer/8-mer complementary complex, most likely through the stacking interaction of the pyran aromatic system with the neighboring nucleotide bases. The effect of chromone (or coumarin) derivatives on the stability of the oligonucleotide complexes (delta delta G at 37 degrees C ranged from -1.0 to -1.7 kcal/mol) was shown to be comparable to the effect of one nucleotide base pair and similar to the effect (delta delta G at 37 degrees C ranged from -1.5 to -2.0 kcal/mol) found for acridineoligonucleotide conjugates served in this study as a reference.

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A new strategy based on the use of cooperative tandems of short oligonucleotide derivatives (TSOD) has been proposed to discriminate a "right" DNA target from a target containing a single nucleotide discrepancy. Modification of a DNA target by oligodeoxyribonucleotide reagents was used to characterize their interaction in the perfect and mismatched complexes. It is possible to detect any nucleotide changes in the binding sites of the target with the short oligonucleotide reagent. In the presence of flanking di-3',5'-N-(2-hydroxyethyl)phenazinium derivatives of short oligonucleotides (effectors) the tetranucleotide alkylating reagent modifies DNA target efficiently and site-specifically only in the perfect complex and practically does not modify it in the mismatched complex. It has been shown that TSOD is much more sensitive tool for the detection of a point mutation in DNA as compared to a longer oligonucleotides.

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