Water-elutability of nucleic acids from metal-chelate affinity adsorbents: enhancement by control of surface charge density.
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 several-fold by addition of 20 volume% 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 acid 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 is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20 mM HEPES, 240 mM NaCl, pH 7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.
Fu
Joseph Y
JY
Department of Chemical Engineering, University of Houston, Houston, TX 77204-4004, USA.
Potty
Ajish S R
AS
Fox
George E
GE
Willson
Richard C
RC
D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity; D003300:Copper / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007098:Imino Acids / Q000737:chemistry; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry* / Q000302:isolation & purification; D011687:Purines / Q000737:chemistry; D012331:RNA, Fungal / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry; D013499:Surface Properties; D014867:Water / Q000737:chemistry*
D002614:Chelating Agents; D007093:Imidazoles; D007098:Imino Acids; D008024:Ligands; D009841:Oligonucleotides; D011687:Purines; D012331:RNA, Fungal; D014867:Water; D003300:Copper; C029899:imidazole; D012685:Sepharose; C008109:iminodiacetic acid