Site-specific laser modification (cleavage) of oligodeoxynucleotides

Sequence-specific photomodification of oligodeoxynucleotide pAGAGTATTGACTTA ("a target") has been carried out with the aid of complementary fluorescent probes. Such a probe consisted of oligodeoxynucleotide pAATACTCT and a chromophore group attached to its 5' end. Three different derivatives of ethidium bromide were used as a chromophore. The photomodification was induced by nitrogen laser radiation (337 nm, 15 MW/cm2). The irradiation induces the following photodamages: target cleavage at the specific binding site with a cutting off of the 8-mer from its 5' end (yield up to 12%), formation of specific covalent adduct target-probe with a yield of 20-70%, and piperidine-sensitive target modifications with a 7-27% yield (for different chromophores). The total yield of specific photodamages of all kinds is 50-80%. The target cleavage and generation of piperidine-sensitive modifications are optically nonlinear processes. Piperidine treatment of the irradiated samples led to specific cleavage of the target with the yield up to 40%. All kinds of observed modifications are not influenced by high concentrations of free radical scavengers: 1.3M tBuOH and 10 mM cystamine. The pattern of cleavage indicates that the most probable position of the chromophore is between T8 and G9 of the target, i.e., the chromophore stacks on top of the last A.T base pair of the duplex. The aggregate of evidence is in agreement with the mechanism of nonlinear photomodification (the cleavage and generation of piperidine-sensitive modifications) based on the transfer of two-photon excitation energy from the chromophore to the target.