Nanopore-Based Profiling of PEGylation in Nucleic Acid Therapeutics

Area of Science:

• Biotechnology
• Nanotechnology
• Therapeutic Drug Development

Background:

• Nucleic acid therapeutics (NATs) show promise for targeted disease treatment.
• Polyethylene glycol (PEG) conjugation improves NAT stability and circulation time.
• Current methods struggle to accurately characterize PEG heterogeneity in NATs.

Purpose of the Study:

• To develop and apply nanopore sensing for precise quantification of PEG conjugation efficiency on NATs.
• To assess the stability and PEGylation of the RNA aptamer pegaptanib in a serum background.
• To demonstrate nanopore sensing's capability to resolve PEG moieties of varying molecular weights.

Main Methods:

• Assembly of DNA nanostructures for binding the RNA aptamer pegaptanib.
• Utilizing solid-state nanopores for single-molecule quantification of pegaptanib PEGylation.
• Evaluating pegaptanib PEGylation and stability in a complex serum environment.

Main Results:

• Nanopore sensing accurately quantified PEG conjugation efficiency on pegaptanib.
• The method successfully resolved PEG moieties with distinct molecular weights.
• Pegaptanib PEGylation and stability were assessed in a serum background.

Conclusions:

• Single-molecule nanopore sensing offers a powerful tool for characterizing polymer-RNA conjugates.
• This technique enables iterative improvements in oligonucleotide design for NATs.
• Nanopore sensing provides a direct method for assessing NAT stability in biological fluids, advancing NAT development.