Nanopore-Based Profiling of PEGylation in Nucleic Acid Therapeutics.

Nucleic acid therapeutics (NATs), including aptamers, offer effective strategies for programmable and targeted disease treatment. To improve their stability and circulation time, oligonucleotides are often conjugated to hydrophilic polymers, such as polyethylene glycol (PEG). However, current bulk techniques fail to resolve PEG heterogeneity, especially in complex biological environments. Here, we use nanopore sensing to quantify the PEG conjugation efficiency of the FDA-approved RNA aptamer pegaptanib. We assembled DNA nanostructures that bind pegaptanib, and then we used solid-state nanopores to quantify pegaptanib PEGylation. We further assessed pegaptanib PEGylation and stability in a serum background and demonstrated that nanopore sensing resolves PEG moieties of distinct molecular weights within the oligonucleotide conjugates. Single-molecule profiling of polymer-RNA conjugates enables iterative improvements in oligonucleotide design and provides a direct means to assess their stability in complex biological environments, thereby advancing the development of more effective NATs.