Format

Send to

Choose Destination
Nucleic Acids Res. 2015 Mar 11;43(5):2927-45. doi: 10.1093/nar/gkv143. Epub 2015 Feb 20.

Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages.

Author information

1
Department of Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA Lliang@isisph.com.
2
Department of Core Antisense Research, ISIS Pharmaceuticals, Carlsbad, CA 92010, USA.

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.

PMID:
25712094
PMCID:
PMC4357732
DOI:
10.1093/nar/gkv143
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center