Multivalent Peptide-DNA Conjugates Facilitate DNA Internalization in Cells Expressing Glycosaminoglycans

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Delivery

Background:

• Nucleic acids (NAs) show therapeutic promise but face cell internalization challenges.
• Multivalent ligand displays, like GalNAc-NA conjugates, enhance cell-specific NA delivery.
• Glycosaminoglycans (GAGs) on cell surfaces are potential targets for NA delivery, associating with cell-penetrating peptides (CPPs).

Purpose of the Study:

• To explore GAGs as cell-surface handles for CPP-mediated, GAG-dependent internalization of nucleic acids.
• To design and evaluate multivalent displays of GAG-targeting ligands for enhanced DNA internalization.
• To investigate the role of multivalency and develop methods for handling CPP-DNA conjugates.

Main Methods:

• Designed tetravalent CPP-DNA conjugates using the Tat CPP.
• Assessed GAG recognition of monovalent vs. tetravalent conjugates in GAG-expressing cell lines.
• Developed solvation methods to address aggregation issues for purification and evaluation.
• Utilized flow cytometry and confocal microscopy to study internalization mechanisms and GAG engagement.

Main Results:

• Tetravalent CPP-DNA conjugates exhibited significantly higher GAG recognition than monovalent conjugates or unmodified DNA.
• Multivalency was crucial for effective cell-surface GAG binding.
• Solvation methods enabled purification and biological evaluation of the conjugates.
• Internalization was confirmed to be GAG-mediated endocytosis, influenced by multivalency and solvation.

Conclusions:

• Tetravalent CPP-ssDNA conjugates are a promising platform for GAG-directed nucleic acid uptake.
• Multivalency is key for efficient GAG binding and subsequent internalization.
• Developed solvation techniques are vital for the practical application of these conjugates.
• This strategy holds broad implications for precision medicine through targeted NA delivery.