Binding and elution behavior of small deoxyribonucleic acid fragments on a strong anion-exchanger multimodal chromatography resin

•Separation of small single- and double-stranded DNA molecules.•Base-specific separation of single-stranded DNA molecules.•Mixed-mode interactions essential for separation of small nucleic acids.

The separation behavior of small single-stranded from double-stranded DNA molecules has been determined on a multimodal (mixed-mode) chromatography system. The resin used is a strong anion exchanger which also modulates hydrophobic recognition. The intrinsic differences between single- and double-stranded DNAs concerning charge, hydrophobicity and three-dimensional structure render this form of MMC suitable for separation of the different nucleic acid molecules. All DNAs tested bound strongly to the resin and they could be eluted with increasing NaCl concentrations. Each homopolymeric ssDNA sample resulted in a base-specific elution pattern when using a linear NaCl gradient. The elution order was poly(dA) < poly(dC) < poly(dG) < poly(dT) and this order was dependent on the secondary structure of the molecule. Such differences were not observed for small double-stranded DNAs. Due to the more hydrophobic nature of single-stranded DNA molecules they could be separated from double-stranded DNAs.