We have analyzed and compared the molecular structures and dynamics of DNA duplexes containing a nick or a gap of one nucleotide where the base in front of the gap is a guanine. The continuous strand has the sequence 5'(CAGAGTCXCTGGCTC) where the residue X is absent for the nick, 14-mer, and where it is a G residue for the gap. Duplexes were formed with the two corresponding 7-mers. Neither of these is phosphorylated adjacent at the nick site, but it is a good model for a single strand break. For the nick structure, the quantitative NMR data show that the global conformation is very close to canonical B-form DNA, but it displays enhanced local flexibility. For the gap structure, we observe only one species in which the extra G is well stacked into the helix. The two half-helices around this residue also show a B-form conformation. As with the nick duplex, the adjacent G imino protons show enhanced exchange with solvent. The gap does not close completely. Using distance constraints, MD calculations show that the nick conformation is very close to a duplex with no lesion but is indeed more flexible in the central part. The gapped structure shows two families of conformations. One is close to B-DNA, the other is significantly kinked at the gap which reduces the size of the cavity. We observe a spine of hydration within the cavities, similar, but of different geometry in the two cases.
In an attempt to target short purine sequences in view of pharmacological application, we have synthesized three new TFO (triple-helix-forming oligonucleotide) conjugates in which an intercalating oxazolopyridocarbazole (OPC) chromophore is linked by a pentamethylene linker to a 7-mer oligonucleotide matching the polypurine/polypyrimidine sequence located in the HIV-1 U3 LTR end region. The TFO moiety of conjugates are 5'CCTTCCC, 5'GGGAAGG, and 5'GGGTTGG. Their ability to bind to double-stranded DNA targets was examined. This binding is demonstrated by a footprinting technique using DNase I as a cleaving agent. The complex involved intermolecular pyr-pur*pyr or pur-pur*pyr triple helix. Pyrimidine TFO-OPC binds in a pH-dependent manner, whereas the others do not. The formation of the complex has been investigated at neutral pH and increasing temperature. We observed that the protection due to the purine and mixed TFO-OPC was pH independent and remained identical up to 40 degrees C. To determine the position of the OPC chromophore, molecular modeling was undertaken on the purine-conjugate/target complex. It has been suggested that the complex involved the intercalation of the OPC at the triplex-duplex junction with a small unwinding at the next excluded site.
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