title: Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.
abstract: Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms.
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA Lliang@isisph.com.
authors.forename: Xue-hai
authors.initials: XH
authors.lastname: Liang
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Shen
authors.affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D017306:Annexin A2 / Q000235:genetics / Q000378:metabolism; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D000090243:Nucleophosmin; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C472822:ANXA2 protein, human; D017306:Annexin A2; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C574674:La protein, human; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D011506:Proteins; D000090243:Nucleophosmin; D012313:RNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen