(675a) Combinatorial Screening of Ligand-Functionalized Microbeads for Unmethylated and Methylated DNA Binding Application

Nucleic acids (such as DNA, RNA) are foundation for many downstream bioprocessing applications of gene therapies and vaccinations. Also DNA methylation patterns play a key role in evaluating gene expression in various diseases like cancer. Hence enrichment of nucleic acid become a challenging area of research where researchers are using different approaches to improve the process development and improve the recovery rate. Competing technologies to isolate methylated plasmids or respective gene of interest involve antibodies or designing DNA-binding proteins which are mainly geared towards research-scale applications; are time consuming and also expensive. Consequently, new approaches which leads to 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 DNA molecules and microbeads. These parental microbeads were further modified with different amino acids, chemotherapeutic drug ligands in order to obtain a diverse library of ligand-modified microbeads. The library of ligand-modified microbeads was screened for binding unmethylated and methylated DNA in order to identify lead candidates that showed preferential binding to methylated DNA 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. Higher degree (~70%) of pDNA elution was obtained after optimizing mixed buffer conditions with salt additives. Computational study with a novel DNA-Pixel (DIXEL) approach was used to demonstrate amino acid ligand interaction with different regions of methylated or unmethylated pDNA. The DIXEL approach allows for the evaluation of DNA sequences using a 2D representation and study its region-specific ligand interactions based on physical properties like electrostatic potential (EP) and lipophilicity. Our results conclude molecular engineering of a chemically diverse ligand-modified microbead platform; that can lead to identification of novel ligands for enriching methylated or unmethylated DNA for applications in bioseparation and rapid detection of diseases at low-resource settings.