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Development of Gene‐Targeted Polypyridyl Triplex‐Forming Oligonucleotide Hybrids
Author(s) -
Zuin Fantoni Nicolò,
McGorman Bríonna,
Molphy Zara,
Singleton Daniel,
Walsh Sarah,
ElSagheer Afaf H.,
McKee Vickie,
Brown Tom,
Kellett Andrew
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000408
Subject(s) - nuclease , oligonucleotide , dna , restriction enzyme , nucleic acid , genome editing , cleavage (geology) , click chemistry , endonuclease , combinatorial chemistry , chemistry , biology , gene , computational biology , crispr , genetics , paleontology , fracture (geology)
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 chemistry‐based DNA‐cleavage system comprising an artificial metallo‐nuclease (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‐of‐interest 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.