Development of Gene-Targeted Polypyridyl Triplex-Forming Oligonucleotide Hybrids

Nicolò Zuin Fantoni, Bríonna McGorman, Zara Molphy, Daniel Singleton, Sarah Walsh, Afaf H. El-Sagheer, Vickie McKee, Tom Brown, Andrew Kellett*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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 CuII 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.

Original languageEnglish
JournalChemBioChem
Volume21
Issue number24
Pages (from-to)3563-3574
ISSN1439-4227
DOIs
Publication statusPublished - 11. Dec 2020

Keywords

  • chemical nuclease
  • click chemistry
  • copper
  • DNA oxidation
  • triplex-forming oligonucleotides

Fingerprint Dive into the research topics of 'Development of Gene-Targeted Polypyridyl Triplex-Forming Oligonucleotide Hybrids'. Together they form a unique fingerprint.

Cite this