DNA oligonucleotides encoding suppressor tRNA induce readthrough of premature termination codon

Nonsense mutation is a type of DNA mutation that converts sense codon in mRNA into premature termination codon (PTC), causing protein truncation. Recent studies have revealed that suppressor tRNAs, which read the stop codon instead of the natural codon and encode the desired amino acid, induce PTC readthrough and restore protein function. However, RNA is prone to degradation, presenting numerous challenges for tissue delivery in gene therapy. Here, we show that DNA oligonucleotides encoding suppressor tRNA sequences but without additional Pol III promoters induce PTC readthrough in cultured cells. Sequence-matched microRNAs specifically inhibited PTC readthrough mediated by the transfected DNA oligonucleotides. Furthermore, treatment with an RNA Pol III inhibitor significantly reduced DNA oligonucleotide-mediated PTC readthrough. These findings support the conclusion that functional suppressor tRNAs are transcribed from DNA oligonucleotides in cultured cells. These results suggest that DNA oligonucleotides hold potential as a novel therapeutic approach enabling suppressor tRNA expression for nonsense mutation repair.