390231 Aminoglycoside Antibiotic Based Novel Anionic Exchange Resins for Plasmid DNA Binding and Purification
Plasmid DNA is playing a decisive role as a therapeutic option for multiple diseases such as cancer, cystic fibrosis and as DNA vaccines. Future clinical treatments that use plasmid DNA must be able to produce pharmaceutical grade quality of DNA in large amounts. Currently, plasmid DNA is grown in bacterial broths from which is extracted by cell lysis and further downstream processing etc. Although significant techniques have been created for downstream processing and extraction of plasmid DNA in large amounts, there is a significant room for improvement.
We report a novel aminoglycoside based hydrogel microresins that show a very high binding capacity to plasmid DNA. Natural ability of aminoglycosides to bind to nucleic acids was exploited in allowing increased binding to plasmid DNA. Aminoglycoside was converted into hydrogel microbeads using emulsion based polymerization. ~10 um microbeads were found to bind 45 ug of plasmid DNA/mg of beads at a concentration of 300 mg/L of plasmid DNA using a Langmuir adsorption isotherm. To increase the efficiency of plasmid DNA binding, the amines were quaternized using GTMAC (Glycidyl trimethyl ammonium chloride). Quaternized microbeads were found to bind almost 300 ug of plasmid DNA/mg of beads at a concentration of 200 mg/L of plasmid DNA, almost binding 30% of plasmid DNA of their weight.
Confocal microscopy was used to study the location of plasmid DNA binding on to the beads. It was noted that the DNA bound majorly at the surface on the beads. To desorb the pDNA, 1M sodium chloride was enough to desorb almost 90% of bound pDNA from unquaternized beads whereas only 35% from quaternized beads. Recovery of plasmid DNA was greatly improved when 1.25 M sodium chloride solution supplemented with 15% isopropanol at 50C was used. The buffer was sufficient to desorb 75% of adsorbed plasmid DNA from the quaternized beads. Around 200 ug of plasmid DNA was recoverable from 1 mg of the quaternized beads.
Unquaternized beads were also found useful in in-situ cancer cell lysis and DNA extraction and binding. Hence, we show a new microresin that can significantly enhance the binding towards plasmid DNA in a reliable way. Our aminoglycoside microbead platform can also be easily modified with conjugation of multiple ligands that can provide selectivity to the resin. We believe aminoglycoside microbeads provide a multipurpose material that can be used towards different applications related but not limited to plasmid DNA.