A Click Chemistry Approach to Developing Molecularly Targeted DNA Scissors

Abstract

Nucleic acid click chemistry was used to prepare a family of chemically modified triplex forming oligonucleotides (TFOs) for application as a new gene-targeted technology. Azide-bearing phenanthrene ligands-designed to promote triplex stability and copper binding-were 'clicked' to alkyne-modified parallel TFOs. Using this approach, a library of TFO hybrids was prepared and shown to effectively target purine-rich genetic elements in vitro. Several of the hybrids provide significant stabilisation toward melting in parallel triplexes (>20 °C) and DNA damage can be triggered upon copper binding in the presence of added reductant. Therefore, the TFO and 'clicked' ligands work synergistically to provide sequence-selectivity to the copper cutting unit which, in turn, confers high stabilisation to the DNA triplex. To extend the boundaries of this hybrid system further, a click chemistry-based di-copper binding ligand was developed to accommodate designer ancillary ligands such as DPQ and DPPZ. When this ligand was inserted into a TFO, a dramatic improvement in targeted oxidative cleavage is afforded.