Sequence- and Base-Selective Modification of mRNA within and nearby Premature Termination Codon and Its Effect on the...

Single-nucleotide variants represent a major class of disease-causing mutations. Among them, nonsense mutations generate premature termination codons (PTCs), resulting in truncated proteins that are nonfunctional or toxic. Because PTCs are responsible for numerous inherited disorders and cancers, various strategies to promote translational readthrough of PTCs have attracted considerable therapeutic interest. However, no clinically effective strategy is currently available. A major limitation of small-molecule readthrough agents is their inability to discriminate between PTCs and natural termination codons, raising concerns about the unintended readthrough of normal stop codons. Therefore, novel mechanism-based strategies capable of selectively promoting PTC readthrough while preserving normal translation termination are highly desirable. In our previous study, we demonstrated that functionality-transfer oligonucleotides (FT-Probes) enabled selective modification of the adenosine residue within a PTC, thereby promoting efficient translational readthrough and production of full-length peptides in an Escherichia (E.) coli translation system. In this study, we investigated the applicability of this approach in a human cell-free translation system. FT-probes were employed to selectively modify adenosine or cytidine residues within or adjacent to a PTC, and the resulting effects on translation were evaluated. In contrast to the efficient readthrough observed in the E. coli translation system, FT-Probe-mediated modification produced only limited effects on the translational readthrough in the human cell-free translation system, indicating substantial mechanistic differences in the recognition of chemically modified PTCs between bacterial and human translational machinery.