Efficient suppression of premature termination codons with alanine by engineered chimeric pyrrolysine tRNAs.

Autor:	Awawdeh A; Department of Molecular Biology and Biophysics, University of Connecticut School of Medicine, 263 Farmington Avenue, Farmington, CT 06030, USA., Tapia A; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.; Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA., Alshawi SA; Department of Genetics and Genome Sciences, Institute for Systems Genomics, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06030, USA., Dawodu O; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA., Gaier SA; Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 64 Turner Street, London, E1 2AD, UK., Specht C; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA., Beaudoin JD; Department of Genetics and Genome Sciences, Institute for Systems Genomics, University of Connecticut Health Center, 400 Farmington Avenue, Farmington, CT 06030, USA., Tharp JM; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA., Vargas-Rodriguez O; Department of Molecular Biology and Biophysics, University of Connecticut School of Medicine, 263 Farmington Avenue, Farmington, CT 06030, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2024 Nov 18. Date of Electronic Publication: 2024 Nov 18.
DOI:	10.1093/nar/gkae1048
Abstrakt:	Mutations that introduce premature termination codons (PTCs) within protein-coding genes are associated with incurable and severe genetic diseases. Many PTC-associated disorders are life-threatening and have no approved medical treatment options. Suppressor transfer RNAs (sup-tRNAs) with the capacity to translate PTCs represent a promising therapeutic strategy to treat these conditions; however, developing novel sup-tRNAs with high efficiency and specificity often requires extensive engineering and screening. Moreover, these efforts are not always successful at producing more efficient sup-tRNAs. Here we show that a pyrrolysine (Pyl) tRNA (tRNAPyl), which naturally translates the UAG stop codon, offers a favorable scaffold for developing sup-tRNAs that restore protein synthesis from PTC-containing genes. We created a series of rationally designed Pyl tRNAScaffold Suppressor-tRNAs (PASS-tRNAs) that are substrates of bacterial and human alanyl-tRNA synthetase. Using a PTC-containing fluorescent reporter gene, PASS-tRNAs restore protein synthesis to wild-type levels in bacterial cells. In human cells, PASS-tRNAs display robust and consistent PTC suppression in multiple reporter genes, including pathogenic mutations in the tumor suppressor gene BRCA1 associated with breast and ovarian cancer. Moreover, PTC suppression occurred with high codon specificity and no observed cellular dysregulation. Collectively, these results unveil a new class of sup-tRNAs with encouraging potential for tRNA-based therapeutic applications. (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu