Applications of charge clustering and surface charge manipulation in biomolecule adsorption

Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased up to ten-fold by addition of 20 volume percent of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acids adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability at the cost of half of the binding affinity is demonstrated for baker's yeast RNA bound to 10% Cu(II)-underloaded IDA Chelating Sepharose in a binding buffer of 20mM HEPES, 240mM NaCl, pH 7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.;We also examined the possibility of improving ion-exchange adsorbent performance by nanoscale structuring of ligands into clusters of fixed size rather than a random distribution of individual charges. The calcium-depleted form of the protein alpha-lactalbumin, which displays a cluster of acidic amino acid residues, showed up to 14-fold enhanced adsorption affinity on clustered-charge pentalysine-amide and pentaarginine-amide adsorbents as compared to single-charge lysine-amide and arginine-amide adsorbents of matched total charge. Two differently charge-clustered mutants of rat microsomal cytochrome b5, El 1Q and E44Q, with the same total charge also were well differentiated by clustered-charge adsorbents, with adsorption affinity enhanced by 2.2 fold going from dispersed to clustered. Clustered pentaarginine-amide with 2.8 mM total charge shows higher affinity and capacity than Q Sepharose with 215 mM total charge. Thus, an organized rather than random distribution of charges may produce adsorbents with higher capacity and selectivity, especially for biomolecules with inherent charge clustering (e.g., nucleic acids).