Oligonucleotides and nucleic acid analogues that alter gene expression are showing therapeutic promise for selected human diseases. The modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, however, such modifications may also confer unexpected physicochemical and biological properties. Here we report that transfection of cultured cells with 2′ O-methyl phosphorothioate antisense sequences resulted in numerous novel large nuclear inclusions in the form of highly structured fibril-like aggregates that co-stained for the paraspeckle proteins, NONO, SFPQ, PSPC1 and FUS. Other nuclear proteins showed altered distribution in 2′ O-methyl phosphorothioate transfected cells. Intranuclear inclusions begin to form within four hours of transfection, and become dominant structures throughout the nucleus within 24 hours. The inclusions appear stable once formed and may remain evident on the culture substrate, even after death and disintegration of the cell. Transmission electron microscopy on transfected cells revealed numerous large, regular structures reminiscent of amyloid deposits, with electron dense regions. Furthermore, gene ontology analysis following RNA sequencing demonstrated significant disruptions to chromatin silencing; regulation of autophagy; nucleotide excision repair; membrane and organelle organization; apoptosis; signaling and protein transmembrane transport, following 2′ O-methyl phosphorothioate transfection. Sequences on a phosphodiester backbone and phosphorodiamidate morpholino oligomers elicited no such consequences. These data suggest that the toxic effects and adverse events reported after clinical evaluation of phosphorothioate nucleic acid drugs may be mediated, at least in part, by non-specific interaction of nuclear components with the phosphorothioate backbone.