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.

Oligonucleotides bearing an aliphatic amino group at the C5-position of deoxyuridine (ULNH2TCCCA, TULNH2CCCA, ULNH2CCACTT, where L = -CH2-, -CH2OCH2CH2- or -CH2NHCOCH2CH2-) have been synthesized. The photoactive (p-azidotetrafluorobenzamido, 2-nitro-5-azidobenzamido, or p-azidobenzamido), alkylating [4-[N-(2-chloroethyl)-N-methylamino]benzyl], or intercalating [N-(2-hydroxyethyl)phenazinium] groups were attached to the amino linker of oligonucleotides. The Tm values were determined for the duplexes formed by the above oligonucleotide derivatives. The alkylating group does not change the melting temperature of the corresponding duplex. The duplex stability is increased a little in the case of photoactive groups. The influence of the phenazinium residue on the duplex stability strongly depends on its location in the oligonucleotide. The spacer length between the C5 atom of deoxyuridine and the photoactive or phenazinium group was shown to influence the complementary duplex stability.

A highly efficient, sequence-specific photomodification of single-stranded (ss) and double-stranded (ds) DNA fragments was carried out with a hexadecathymidilate derivative, R approximately p(T)16 (R-perfluoroarylazido group), using 27-base pair DNA fragments as a target [table: see text] The main points of modification were G7 and G24 of the A-rich strand of the ss target and G7 and G22 of the A-rich and T-rich strands, respectively, for the ds target. The extent of photomodification was 60%-77% for ss DNA and 10%-53% for ds DNA depending on the reaction conditions. Photomodification increased in buffer with a high ionic strength (1.0 M) and at low temperature (4 degrees C) when presumably the triplexes were more stable.

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Highly efficient site-specific photomodification of single-stranded DNA targets was achieved with oligonucleotide reagents bearing aromatic azido groups (R (R1 = p-azidotetrafluorobenzoyl, R2 = 2-nitro-5-azidobenzoyl, R3 = p-azidobenzoyl) at either the terminal phosphate or at the C5 position of deoxyuridine at the end or inside of the oligonucleotide chain. The extent of modification strongly depends on the reagent type. It does not exceed 5% in the case of the reagent with R3. It was 25%-50% and 60%-70% for the reagents with R2 and R1 depending on the target structure. The reagent with perfluoroarylazido group R1 appeared to be most efficient. The extent of covalent adduct formation amounts to 70% for all reagents bearing a perfluoroarylazine group at the end of the oligonucleotide chain, independently of whether it was attached to the 3'- or 5'-phosphate or to the C5 of deoxyuridine. The reagents with the reactive group within the chain provided fewer cross-links (50%-55%). The reagents with R1 and R2 were found to be sensitive to the nucleotide structure of the target. Guanine and cytosine residues were modified preferentially when adjacent to the R1 or R2 group of the reagent, respectively.