PHOSPHOROTHIOATE ANTISENSE OLIGONUCLEOTIDES INDUCE THE FORMATION OF NUCLEAR-BODIES

Antisense oligonucleotides are powerful tools for the in vivo regulati on of gene expression. We have characterized the intracellular distrib ution of fluorescently tagged phosphorothioate oligodeoxynucleotides ( PS-ONs) at high resolution under conditions in which PS-ONs have the p otential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bod ies), which were set against a diffuse nucleoplasmic population exclud ing nucleoli. PS bodies are nuclear structures that formed in cells af ter PS-ON delivery by transfection agents or microinjection but were o bserved irrespectively of antisense activity or sequence. Ultrastructu rally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower fr equency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entit ies that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-O N pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our dat a provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.