Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides

Abstract

Antisense oligonucleotides (ASOs) are versatile tools that can regulate multiple steps of RNA biogenesis in cells and living organisms. Significant improvements in delivery, potency, and stability have been achieved through modifications within the oligonucleotide backbone, sugar and heterocycles. However, these modifications can profoundly affect interactions between ASOs and intracellular proteins in ways that are only beginning to be understood. Here, we report that ASOs with specific backbone and sugar modifications can become localized to cytoplasmic ribonucleoprotein granules such as stress granules and those seeded by the aggregation of specific ASO-binding proteins such as FUS/TLS (FUS) and PSF/SFPQ (PSF). Further investigation into the basis for ASO-FUS binding illustrated the importance of ASO backbone and hydrophobic 2′ sugar modifications and revealed that the C-terminal region of FUS is sufficient to retain ASOs in cellular foci. Taken together, the results of this study demonstrate that affinities of various nucleic acid binding domains for ASO depend on chemical modifications and further demonstrate how ASO–protein interactions influence the localization of ASOs.

Mouse genome-wide association studies and systems genetics uncover the genetic architecture associated with hepatic pharmacokinetic and pharmacodynamic properties of a constrained ethyl antisense oligonucleotide targeting Malat1

“…It is generally accepted that some pathways of internalization and trafficking are productive, i.e leading to a pharmacological effect, while others are pharmacologically non-productive ‘sinks’ [ 41 , 42 ]. Several structural, nucleic acid binding and chaperone proteins have been shown to bind to ASOs and influence intracellular localization and trafficking within the cell [ 42 , 43 ]. Most importantly, some of these interactions are known to impact ASO therapeutic potency [ 4 ].…”

Application of 2′-O-(2-N-Methylcarbamoylethyl) Nucleotides in RNase H-Dependent Antisense Oligonucleotides

“…One of the major mechanisms mediating the hybridization-independent effects is the binding of chemically modified ASOs to plasma and cellular proteins. Introduction of chemical modifications on ASOs is known to result in differential binding affinities to cellular proteins [ 14 , 15 ]. For example, 2′-F-modified oligonucleotides cause rapid degradation of P54nrb and polypyrimidine tract-binding protein-associated splicing factor (PSF) which belong to the Drosophila behavior/human splicing family [ 16 ].…”

RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis

“…Other studies investigated the effects of ASO on the oligonucleotide backbone, sugar, and heterocycles to promote delivery, potency, and stability to target FUS. These studies evidenced that the affinities of nucleic acid binding domains depend on chemical changes and that the interaction between ASO and protein affects the localization of ASOs themselves [92]. These data strongly indicate that ASO-based therapy could be central in treating ALSrelated genes, although there is great attention on the relation between the therapeutic outcomes and the stage of disease progression and on the time of intervention.…”