Development of stapled NONO-associated peptides reveals unexpected cell permeability and nuclear localisation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The non-POU domain-containing octamer-binding protein (NONO) is a nucleic acid-binding protein implicated in various cellular functions and identified as a potential cancer target.
• Understanding the structural motifs mediating NONO interactions, beyond its dimerization capabilities, is crucial for therapeutic development.

Purpose of the Study:

• To investigate structural motifs for NONO binding using macrocyclised helical peptides.
• To explore the potential of these peptides as tools for studying NONO in a cellular context.

Main Methods:

• A stapling approach was used to macrocyclise helical peptides derived from insulin-like growth factor binding protein (IGFBP-3) and the NONO dimerization domain.
• Various chemistries, including Pd-catalysed cross-coupling, cysteine arylation, and alkylation, were employed to enhance peptide helicity.
• Peptide binding to the NONO dimer was assessed, and cell permeability and nuclear localization were observed using dye-labelled peptides and live confocal microscopy.

Main Results:

• Macrocyclisation successfully improved peptide helicity.
• Modest binding of peptides to the NONO dimer was observed, though saturation was not achieved at micromolar concentrations.
• Dye-labelled peptides exhibited unexpected cell permeability and preferential nuclear localization in live cells.

Conclusions:

• Macrocyclised peptides show potential for studying NONO interactions within cells.
• The cell-permeable and nuclear-localizing properties of these peptides open avenues for developing novel peptide-based tools for NONO research.
• Further optimization may be needed to achieve saturated binding for therapeutic applications.