An unnatural enzyme with endonuclease activity towards small non-coding RNAs

Noreen Ahmed, Nadine Ahmed, Didier A. Bilodeau and John Paul Pezacki ()
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Noreen Ahmed: University of Ottawa
Nadine Ahmed: University of Ottawa
Didier A. Bilodeau: University of Ottawa
John Paul Pezacki: University of Ottawa

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: Abstract Endonucleases are enzymes that cleave internal phosphodiester bonds within double-stranded DNA or RNA and are essential for biological functions. Herein, we use genetic code expansion to create an unnatural endonuclease that cleaves non-coding RNAs including short interfering RNA (siRNA) and microRNAs (miRNAs), a function that does not exist in nature. We introduce a metal-chelating unnatural amino acid, (2,2′-bipyridin-5-yl)alanine (BpyAla) to impart endonuclease activity to the viral suppressor of RNA silencing protein p19. Upon binding of copper, the mutant p19-T111BpyAla displays catalytic site-specific cleavage of siRNA and human miRNAs. Catalysis is confirmed using fluorescence polarization and fluorescence turn-on. Global miRNA profiling reveals that the engineered enzyme cleaves miRNAs in a human cell line. The therapeutic potential is demonstrated by targeting miR-122, a critical host factor for the hepatitis C virus (HCV). Unnatural endonuclease function is shown to deplete miR-122 levels with similar effects to an antagomir that reduces HCV levels therapeutically.

Date: 2023
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DOI: 10.1038/s41467-023-39105-0

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