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Profiling the interactome of oligonucleotide drugs by proximity biotinylation

ABSTRACT: Drug-ID applies proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug molecule with a biotin ligase enables targeted biotinylation of the drug-bound proteome and its identification by SILAC-MS/MS. We investigate the interactome of two well-known small molecule drugs, JQ1 and SAHA, and applied it for the identification of the interactome of RNaseH-recruiting antisense oligonucleotides (ASO). Drug-ID allows to de novo profile the drug-protein interactome under native conditions, directly inside living cells and at the pharmacologically effective concentration of the drug using minimal amount of input material. We apply the technology to study the dose-dependent aggregation of ASOs and the effect of different wing chemistries (LNA, Fluoro, MOE) and ASO lengths on the ASO-protein interactome. Finally, we demonstrate the detection of stress-induced interactome changes (Actinomycin D treatment) in living cells. Particularly broadly applicable in the field of oligonucleotide therapeutics is the in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Kidney Cell Line, Cervical Cancer Cell Line

SUBMITTER: Mirita Franz-Wachtel

LAB HEAD: Thorsten Stafforst

PROVIDER: PXD045992 | Pride | 2024-01-09

REPOSITORIES: Pride

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