Combinatorial Discovery of Ligand-Fucntionalized Microbeads for Selective Enrichment of Methylated and Unmethylated DNA

DNA methylation patterns play a vital role in determining gene expression in different diseases including cancer. In addition, therapeutic plasmid DNA (pDNA) molecules, used in non-viral gene therapy, are frequently methylated by the enzymes present in bacterial strains employed in their bio-processing. Current approaches to isolate methylated plasmids and genomic DNA involve antibodies, which are mainly geared towards research-scale applications. Consequently, new approaches that can lead to the enrichment of methylated or unmethylated DNA from biological samples and bioprocessing media, in a rapid and cost-effective manner, can lead to transformative advances in these fields. We have developed aminoglycoside-derived microbeads, approximately 10μm in diameter, for binding DNA based on Coulomb interactions between the beads and DNA molecules. These parental microbeads were modified with different amino acids, lipids, and chemotherapeutic drugs, in order to obtain a diverse library of ligand-modified microbeads. The library of ligand-modified microbeads was screened for binding unmethylated and methylated pDNA (a model plasmid that encodes for luciferase protein) in order to identify lead candidates that showed preferential binding to methylated pDNA over the unmethylated analog or vice versa. Adsorption isotherms were determined for both sets of leads and the data were fit to a Langmuir isotherm model. Elution of adsorpbed DNA was investigated with different buffers and organic modifiers. Finally, quantitative structure activity relationship (QSAR) models were employed to obtain physicochemical insights into molecular interactions that govern ligand binding to methylated as well as unmethylated DNA. In conclusion, our results indicate that molecular engineering of a chemically diverse ligand-modified microbead platform can lead to rapid identification of novel ligands for enriching methylated or unmethylated DNA for applications in bio-manufacturing of gene therapy vectors and in the rapid detection of diseases, including in low-resource settings.