A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [alpha]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[alpha]-thymidylate: a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[alpha]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[alpha]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [alpha]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.

On the basis of the structure of DNA-psoralen bis adducts (formed by psoralen with two thymines on opposite strands), a psoralen-oligonucleotide conjugate was designed to photoinduce a cross-link between the two DNA strands at a specific sequence. Psoralen was attached via its C-5 position to a 5'-thiophosphate group of an 11-mer homopyrimidine oligonucleotide. The 11-mer binds to an 11-base-pair homopurinehomopyrimidine sequence of a DNA fragment, where it forms a triple helix. Upon near-UV-irradiation, the two strands of DNA are crosslinked at the TpA step present at the triplex-duplex junction. The reaction is specific for the homopurine'homopyrimidine DNA sequence and requires both oligonucleotide recognition of the DNA major groove and intercalation of psoralen at the triplex-duplex junction. The yield of the photoinduced cross-linking reaction is quite high (>80%). Such psoralen-oligonucleotide conjugates are probes of sequencespecific triple-helix formation and could be used to selectively control gene expression or to induce site-directed mutations.

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A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.

The three-dimensional structural analysis of DNA undecamer 5'd(C1G2C3A4C5X6C7A8C9G10C11)3', 3'd(G22C21G20T19G18T17G16T15G14C13G12)5' duplex in which the X residue is a modified abasic site [3-hydroxy-2-(hydroxymethyl)tetrahydrofuran] has been performed using NOESY, DQFCOSY, TOCSY, and 31P-1H HSQC-TOCSY spectra in relation with molecular dynamics simulations. A total of 249 distances and 224 dihedral angles were used for construction. The optimal distances were calculated using the complete relaxation matrix method from hybrid matrices which were built with the experimental NOE intensities and additional data derived from either standard A- or B-DNA. Six independent refined structures starting from canonical A- and B-DNA were determined on the basis of the NMR data, and all converged to a single family with average rms deviations below 0.6 A and final NOE Rx factors of 0.055 +/- 0.03. A satisfactory agreement was obtained between measured NOE intensities and those resulting from full relaxation matrix calculations. A single intrahelical form of right-handed DNA duplex is observed; the aromatic base of residue T17 opposite the abasic site is stacked inside the helix. No clear correlation was detected between the C5 and C7 residues, excluding their proximity and the looping out of the abasic site. The abasic site induces a kink of about 30 degrees in the DNA duplex. This kink allows the formation of a bifurcated hydrogen bond between the amino protons of C5 and the O4 oxygen of T17. A detailed analysis of the final structures and their comparison with previous studies of abasic site lesions are described.

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Deoxyribonolactone in DNA is an oxidized abasic site damage that is produced by a variety of physical and chemical agents such as gamma-irradiation and ene-diyne antibiotics. The extent and biological significance of the lesion are poorly documented due to the high lability of the damaged DNA. The chemistry of degradation of deoxyribonolactone-containing DNA was investigated using oligonucleotides of different length (5-, 11-, 23-, 34-mers) in which the lactone was photochemically generated, as already reported, from oligonucleotide precursors containing a photoactive nitroindole residue. The procedure was successfully extended to double-strand synthesis by irradiation of the preformed duplex in which one strand contained the nitroindole residue. The degradation kinetics were investigated as a function of pH, temperature, length, and ionic strength. The cleavage fragments resulting from beta- and delta-eliminations were isolated and identified by (1)H NMR. It was found that the lesion is extremely sensitive to pH and temperature while slightly dependent upon ionic strength, length, and sequence. The cleavage rates for the beta- and delta-elimination steps are of the same order of magnitude. The deoxyribonolactone site leads to greater instability of DNA than the "regular" deoxyribose abasic site.