biostudies-literatureUnknown37(1)Gaci ACellular 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 intMolecular therapy. Nucleic acids102832https://www.ebi.ac.uk/biostudies/studies/S-EPMC12860984biostudies-literatureIdentification and functional validation of intracellular protein partners of phosphorothioate splice-switching oligonucleotides using AP-MS.PMC12860984Salnot VBruce JPedeux RMenchon GGaci AGoyenvalle AfalseIdentification and functional validation of intracellular protein partners of phosphorothioate splice-switching oligonucleotides using AP-MS.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 int2026-01-01T00:00:00Z2026 Mar2026-06-19T04:44:57.935Z2026-06-19T03:07:05.352ZS-EPMC128609844163098810.1016/j.omtn.2026.102832