Double-stranded DNA unzipping: A fundamental and experimental investigation of fluid-induced stress in ultra-high pressure liquid chromatography systems and slalom columns.

Recently reintroduced for the separation of large (> 1 MDa) double-stranded DNA (dsDNA) and RNA biopolymers, slalom chromatography (SC) exploits the entropic elasticity of long nucleic acid polymers subjected to hydrodynamic drag in packed columns. Using a bioinert Slalom Column (4.6 × 300 mm) packed with 2.5μm particles operated with a 40 mM Tris-acetate-EDTA mobile phase at 30 °C, we examine the unzipping of the dsDNA concatemer strands as they undergo repeated coil-to-stretch transitions under intense fluid stress throughout the ultra-high pressure chromatography system (open tubing and contraction geometries) and the Slalom Column (inter-particle volume). To establish a direct link between the fluid-induced stresses and DNA integrity, we combine theoretical descriptions of stress distribution in particulate packed beds, tubings, and contraction geometries with experiments on λ-DNA concatemers formed via hybridization of a short 12-nt 5 [Formula: see text] single-stranded overhangs (sticky ends). Even at the highest flow rate investigated (1.2 mL min-1, 11,000 psi), the maximum forces acting on a 27,491 bp concatemer within the column frit (<1 pN) and the packed bed (<10 pN) remain well below the ∼ 15 pN base-pair unzipping threshold. This demonstrates that the narrow (average diameter 0.52μm), tortuous inter-particle flow paths of the slalom column cannot account for the extensive base-pair cleavage observed at elevated flow rates. Instead, the results are consistent with substantially higher fluid stresses generated upstream of the column along the narrowest flow paths of the ultra-high pressure liquid chromatography (UHPLC) system. Replacing the 100μm i.d. active preheater with a single narrower 40μm i.d. tubing (or with a single wider 180μm i.d. tubing) upstream of the column induces complete (or reduced) dissociation of the 14,140 bp concatemer at flow rates as low as 0.3 mL min-1 (or 1.0 mL min-1), under conditions for which no dissociation (or significant dissociation) was observed with the standard instrument configuration. These findings identify the narrowest pre-column tubing as the primary source of dsDNA unzipping and support maintaining tubing inner diameters larger than 125μm throughout the pre-column flow path when analyzing sensitive and large biomacromolecules, including long nucleic acids, protein assemblies, lipid nano-particles, and supramolecular complexes.