Identification and functional validation of intracellular protein partners of phosphorothioate splice-switching...

Cellular uptake and intracellular distribution of phosphorothioate-modified antisense oligonucleotides (PS-ASOs) are mediated by protein interactions. While several PS-ASOs-binding proteins have been identified, mainly using gapmer designs with 2'-O-methoxyethyl (2'MOE) modifications, less is known about protein partners of splice-switching oligonucleotides (SSOs) with alternative ribose modifications. Here, using affinity purification mass spectrometry (AP-MS), we identified the intracellular protein partners of PS-SSOs of the same sequence with three distinct ribose modifications: tricyclo-DNA (tcDNA), locked nucleic acid (LNA), and 2'MOE. Interestingly, we found previously reported PS interactors, such as GRSF1, NONO, and NCL, as well as uncharacterized protein partners. Four shared interactors identified in this study, ERC1, SPIRE1, THRAP3, and GOLGA2, were selected based on functional relevance and tested for their impact on exon skipping efficacy using PS-SSOs targeting the human Duchenne muscular dystrophy (DMD) transcript. RNA interference-mediated knockdown of each protein led to a reduction of exon skipping efficiency, suggesting that these proteins may contribute to PS-ASOs activity regardless of their sugar modifications. Overall, our results provide a set of intracellular protein interactors of different PS-ASOs, representing a valuable resource to explore mechanisms underlying their activity and offering potential leads for the optimization of oligonucleotide therapeutics.