A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN-TFO hybrids is based on application of the alkyne-azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5'- or 3'-ends or in the middle of the TFO stretch. The diverse hybrids efficiently formed triplexes with the target purine-rich sequence and their copper complexes were studied for their ability to cleave dsDNA in the presence of ascorbate as a reductant. In all cases, the influence of the nature and length of the AMN-TFO, time, conditions and amounts of ascorbate were studied, and optimum conjugates and a procedure that gave reasonably efficient (up to 34 %) cleavage of the target sequence, while rendering an off-target dsDNA intact, were found. The footprint of cleavage on PAGE was identified only in one case, with low conversion; this means that cleavage does not proceed with single nucleotide precision. On the other hand, these AMN-TFO hybrids are useful for the selective degradation of target dsDNA sequences. Future improvements to this design may provide higher resolution and selectivity.
Keywords: DNA cleavage; click chemistry; copper; nucleases; oligonucleotides.
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