Triplex forming oligonucleotides (TFOs) are short strands of nucleotides that can hybridise to duplex DNA and form triple helical structures. As triplex formation is sequence selective, there is significant interest in applying this technology for antigene therapy. In this work, primer extension (PEX) protocols were designed and utilised to enzymatically synthesise TFOs bearing artificial metallo-nucleases (AMNs). Initially, single nucleotide incorporation–primer extension (SNI-PEX) was employed to generate TFOs bearing a single modification. This was then expanded to the novel multiple nucleotide incorporation–primer extension (MNIPEX) protocol, which enabled multiple different modified bases to be incorporated at specific sites in the TFO sequence. Click chemistry was employed to generate a library of functionalised nucleotides, which were then enzymatically incorporated into the TFO strand by a DNA polymerase. These AMN-TFO hybrids can recognise and damage specific DNA sequences, and therefore are capable of targeting a gene of interest. Real-time PCR (qPCR) and polyacrylamide gel electrophoresis (PAGE) protocols were then developed to analyse and compare the activity of these sequence-selective hybrids. PAGE was employed to visualise the sequence specificity of the TFO-drug hybrids and to identify ‘on’ and ‘off’ targeting effects. Next, the DNA damaging capabilities of three distinct TFO-hybrids, each bearing a unique AMN ligand (5N3 -TPMA, 6N3 -TPMA and 4N3 benzyl-DPA), were quantified by a specifically developed qPCR protocol. Here, target DNA post treatment with the AMN-TFO hybrid, was amplified in the presence of SYBR Green I and the resulting fluorescent signal was compared to that of the untreated controls. Results show the AMN-TFOs can discriminate between target and off-target DNA, and differences in the DNA damaging capabilities of each AMN ligand were recorded. These results indicate that TFO-drug hybrids may have the ability to overcome problems associated with non-molecularly targeted DNA damaging agents, and represent a new class of gene knockout agent.