Monolithic spin column desalting: A rapid and size-independent solution for high throughput nucleic acid cleanup

Modern bioprocessing of nucleic acids depends on affinity, hydrophobic interaction chromatography and ion exchange chromatography, which require high concentrations of salt for loading or elution. Consequently, in-process samples can contain high concentrations of salts, which may interfere with analytical techniques used for sample characterization, thus decreasing method accuracy, sensitivity and/or robustness. One example of a highly matrix-dependant analytical technique is capillary gel electrophoresis, which is used for the analysis of integrity, size and isoform composition of nucleic acids. Sample desalting is a crucial step before analysis and currently centrifugal ultrafiltration filters, size exclusion/gel filtration spin columns, solid-phase extraction or precipitation are used for such purpose, but each with limitations. Here we present the development of an innovative platform desalting method using monolithic spin columns and apply it as a sample pretreatment step for analysis of oligonucleotides, plasmid DNA and messenger RNA by capillary gel electrophoresis. Utilizing the binding of nucleic acids at slightly acidic pH onto a multimodal anion-exchanging column and eluting them with a low conductivity buffer at neutral pH, these monolith spin columns can efficiently remove residual salts and small molecules. Under optimal conditions, pretreated samples retain the quality of the initial sample with improved sensitivity at higher processing speed compared to ultrafiltration-based desalting methods. One device and one general procedure can be used for rapid desalting of various nucleic acids (oligonucleotides, plasmid DNA, messenger RNA), regardless of their size, which is not attainable with centrifugal filters.