HPLC photofingerprinting of conformational peculiarities and transitions in oligonucleotide duplexes.

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RESUMO

Two self-complementary sequence-isomeric decadeoxyribonucleotides were exposed to UV light under conditions in which they assume duplex structures. After that they were analyzed in the denatured state by reversed-phase high-performance liquid chromatography (HPLC). Characterization of the separated photoproducts allowed localization of cyclobutane pyrimidine dimers in the sequences of the modified oligonucleotides. For [d(GGAAATTTCC)]2, which is known to contain in its central part a stretch of rigid B'-conformation with decreased mobility of constituent bases, lower yields of thymine dimers, as compared with that for ordinary B-form [d(CCTTTAAAGG)]2, were found. On the contrary, mixed thymine-cytosine heterodimers generated in the former oligonucleotide demonstrate the increase in photoreactivity of these residues at the B'-B junction. This is probably due to the peculiar conformation adopted by this decanucleotide. Stimulation of B'-B transition, by increasing the temperature before melting, reduced an inhibition of thymine photodimer formation. During the melting of both oligonucleotides yields of all identified photoinduced cyclobutadipyrimidines were reduced. Possible influences of some metal cations on the stability of the B'-form were also studied by this photoprobing technique. The present study demonstrates the feasibility of HPLC photofingerprinting as a new approach for structural analysis of nucleic acids.

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