Structure-guided antisense oligonucleotides selectively modulate frameshifting of a human gene

Programmed -1 ribosomal frameshifting (-1 PRF) is a conserved translational recoding mechanism that expands proteomic diversity and regulates gene expression through RNA structural elements, most notably stimulatory pseudoknots. This mechanism is common in viruses, where it is used to control stoichiometry of viral protein products generated by the host cell to direct viral replication. Despite its biological importance, strategies to selectively modulate frameshifting remain limited. The mammalian retrotransposon-derived gene PEG10 also relies on -1 PRF to produce a fusion protein, gag-pol, which is necessary for reproduction but has also been implicated in neurological diseases. Here, we establish an antisense oligonucleotide (ASO) targeting an RNA structural element as an effective approach to tune PEG10 frameshifting. Using structure prediction, systematic antisense tiling across the PEG10 pseudoknot, and multiple model systems, we identify a discrete functional hotspot within the lower RNA stem that governs frameshift efficiency. ASOs targeting this region selectively suppress gag-pol production with minimal impact on gag, thereby shifting the ratio of protein products in a dose-dependent manner. Mechanistic dissection using RNase H-active and -inactive ASO designs, pre-annealed duplexes, and fluorescence-based subcellular localization supports a predominantly nuclear mode of action in which ASOs engage nascent PEG10 transcripts and bias pseudoknot folding away from the frameshift-competent conformation. Functional effects are conserved between human cell lines and murine models, including neurons, highlighting the generality of this strategy. Together, our results define RNA structural dynamics as a druggable layer of translational regulation and establish antisense modulation of pseudoknot folding as a way to control endogenous frameshifting. This work provides a conceptual and practical framework for targeting recoding-dependent gene products such as PEG10 in disease and suggests broader applicability of structure-directed ASOs to viral and cellular frameshifting elements.