In the field of nucleic acid therapy there is major interest in the development of libraries of DNA-reactive small molecules which are tethered to vectors that recognize and bind specific genes. This approach mimics enzymatic gene editors, such as ZFNs, TALENs and CRISPR-Cas, but overcomes the limitations imposed by the delivery of a large protein endonuclease which is required for DNA cleavage. Here, we introduce a chemistrybased DNA-cleavage system comprising an artificial metallonuclease (AMN) that oxidatively cuts DNA, and a triplex-forming oligonucleotide (TFO) that sequence-specifically recognises duplex DNA. The AMN-TFO hybrids coordinate Cu II ions to form chimeric catalytic complexes that are programmable-based on the TFO sequence employed-to bind and cut specific DNA sequences. Use of the alkyne-azide cycloaddition click reaction allows scalable and high-throughput generation of hybrid libraries that can be tuned for specific reactivity and gene-ofinterest knockout. As a first approach, we demonstrate targeted cleavage of purine-rich sequences, optimisation of the hybrid system to enhance stability, and discrimination between target and off-target sequences. Our results highlight the potential of this approach where the cutting unit, which mimics the endonuclease cleavage machinery, is directly bound to a TFO guide by click chemistry.

Limitations of clinical platinum(II) therapeutics include systemic toxicity and inherent resistance.M odern approaches,t herefore,s eek new ways to deliver active platinum(II) to discrete nucleic acid targets.I nt he field of antigene therapy, triplex-forming oligonucleotides (TFOs) have attracted interest for their ability to specifically recognise extended duplex DNAtargets.Here,wereport aclickchemistry based approach that combines alkyne-modified TFOs with azide-bearing cis-platinum(II) complexes-based on cisplatin, oxaliplatin, and carboplatin motifs-to generate al ibrary of Pt II -TFO hybrids.T hese constructs can be assembled modularly and enable directed platinum(II) crosslinking to purine nucleobases on the target sequence under the guidance of the TFO.B yc ovalently incorporating modifications of thiazole orange-a knownD NA-intercalating fluorophore-into Pt II -TFOs constructs,e nhanced target binding and discrimination between target and off-target sequences was achieved.

Limitations of clinical platinum(II) therapeutics include systemic toxicity and inherent resistance.M odern approaches,t herefore,s eek new ways to deliver active platinum(II) to discrete nucleic acid targets.I nt he field of antigene therapy, triplex-forming oligonucleotides (TFOs) have attracted interest for their ability to specifically recognise extended duplex DNAtargets.Here,wereport aclickchemistry based approach that combines alkyne-modified TFOs with azide-bearing cis-platinum(II) complexes-based on cisplatin, oxaliplatin, and carboplatin motifs-to generate al ibrary of Pt II -TFO hybrids.T hese constructs can be assembled modularly and enable directed platinum(II) crosslinking to purine nucleobases on the target sequence under the guidance of the TFO.B yc ovalently incorporating modifications of thiazole orange-a knownD NA-intercalating fluorophore-into Pt II -TFOs constructs,e nhanced target binding and discrimination between target and off-target sequences was achieved.

Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators—DPQ and DPPZ—and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.