Patino Guillen, G. | Search bioRxiv

bioRxiv2025

Nanopore-based profiling of PEGylation in nucleic acid therapeutics

Nucleic acid therapeutics, 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 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 assess pegaptanib PEGylation and stability in a serum background and demonstrate that nanopore sensing resolves PEG moieties of distinct molecular weights within 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 nucleic acid therapeutics.

bioengineering↗

bioRxiv2023

Nanopore translocation reveals electrophoretic force on A- and B-form nucleic acids

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of non-canonical RNA:DNA hybrids in electrophoretic transport with the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a non-canonical helix with distinct transport properties from B-form DD molecules. We find RD and DD molecules with the same contour length move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same within experimental errors. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and molecular understanding of electrophoretic transport.

biophysics↗