Nanopore translocation reveals electrophoretic force on non-canonical RNA:DNA double helix

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.