Richard C. Willson, University of Houston, CHEE 4004 4800 Calhoun Rd., Houston, TX 77004-4004
The speaker will attempt to convey his (and the community's) grateful appreciation of Ed Lightfoot. Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have been developing its application to nucleic acids through interactions involving exposed purine bases in single-stranded RNA and oligonucleotides and mis-renatured genomic DNA. We have found that the inclusion of moderate quantities of neutral solutes (e.g., n-propanol) substantially enhances the binding affinity of nucleic acids for IMAC adsorbents. Here we report that RNA can be eluted from IMAC matrices using water with no imidazole competitor by manipulation of adsorbent surface charge, and/or by manipulation of additive concentration. Promotion of hydrolytic hydroxide complexes and deliberate underloading of the negatively charged chelating ligands were used to increase the negative surface charge density of the surface, enhancing repulsive interactions with anionic DNA/RNA at low ionic strength.