Selective hybridization and capture of KRAS DNA from plasma and blood using ion-tagged oligonucleotide probes coupled to magnetic ionic liquids.

Detection of circulating tumor DNA (ctDNA) presents several challenges due to single-nucleotide polymorphisms and large amounts of background DNA. Previously, we reported a sequence-specific DNA extraction procedure utilizing functionalized oligonucleotides called ion-tagged oligonucleotides (ITOs) and disubstituted ion-tagged oligonucleotides (DTOs). ITOs and DTOs are capable of hybridizing to complementary DNA for subsequent capture by a magnetic ionic liquid (MIL) through hydrophobic interactions, π-π stacking, and fluorophilic interactions. However, the performance of the ITOs and DTOs in complex sample matrices has not yet been evaluated. In this study, we compare the amount of KRAS DNA extracted using ITO and DTOs from saline, 2-fold diluted plasma, 10-fold diluted plasma, and 10-fold diluted blood. We demonstrate that ITO/DTO-MIL extraction is capable of selectively preconcentrating DNA from diluted plasma and blood without additional sample preparation steps. In comparison, streptavidin-coated magnetic beads were unable to selectively extract DNA from 10-fold diluted plasma and 10-fold diluted blood without additional sample clean-up steps. Significantly more DNA could be extracted from 2-fold diluted plasma and 10-fold diluted blood matrices using the DTO probes compared to the ITO probes, likely due to stronger interactions between the probe and MIL. The ability of the DTO-MIL method to selectively preconcentrate small concentrations of DNA from complex biological matrices suggests that this method could be beneficial for ctDNA analysis.