Browsing by Author "Alshawi, Sarah A."

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    An engineered glutamic acid tRNA for efficient suppression of pathogenic nonsense mutations
    (Oxford University Press, 2025) Specht, Caitlin; Tapia, Alejandro; Penrod, Sarah; Soriano, Gabriela A.; Awawdeh, Aya; Alshawi, Sarah A.; White, Cody A.; Beaudoin, Jean-Denis; Doud, Emma H.; Vargas-Rodriguez, Oscar; Huang, Yunjie; Tharp, Jeffery M.; Biochemistry and Molecular Biology, School of Medicine
    Nonsense mutations that introduce premature termination codons (PTCs) into protein-coding genes are responsible for numerous genetic diseases; however, there are currently no effective treatment options for individuals affected by these mutations. One approach to combat nonsense-related diseases relies on the use of engineered suppressor transfer RNAs (sup-tRNAs) that facilitate translational stop codon readthrough, thereby restoring full-length protein synthesis. While several sup-tRNAs have shown promising results in preclinical models, many exhibit low PTC suppression efficiency, precluding their use as therapeutics. For example, glutamic acid (Glu) codons represent one of the most common sites for nonsense mutations, yet existing sup-tRNAs are ineffective at suppressing Glu-to-Stop mutations. To address this limitation, here we describe a rationally designed sup-tRNA (tRNAGluV13) with greatly improved ability to suppress PTCs occurring at Glu codons. We demonstrate that tRNAGluV13 efficiently restores protein synthesis from multiple nonsense-containing reporter genes, faithfully installing Glu in response to PTCs. Additionally, we demonstrate that tRNAGluV13 can functionally rescue pathogenic PTCs that cause hereditary breast and ovarian cancer syndrome and cystic fibrosis. The ability of tRNAGluV13 to effectively suppress one of the most common PTC mutations should greatly expand the potential of sup-tRNA-based therapeutics.
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    Efficient suppression of premature termination codons with alanine by engineered chimeric pyrrolysine tRNAs
    (Oxford University Press, 2024) Awawdeh, Aya; Tapia, Alejandro; Alshawi, Sarah A.; Dawodu, Olabode; Gaier, Sarah A.; Specht, Caitlin; Beaudoin, Jean-Denis; Tharp, Jeffery M.; Vargas-Rodriguez, Oscar; Biochemistry and Molecular Biology, School of Medicine
    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.