Water-elutability of nucleic acids from metal-chelate affinity adsorbents: Enhancement by control of surface charge density

Joseph Y. Fu, Ajish S.R. Potty, George E. Fox, Richard C. Willson

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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, pH7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.

Original languageEnglish (US)
Pages (from-to)348-353
Number of pages6
JournalJournal of Molecular Recognition
Volume19
Issue number4
DOIs
StatePublished - Jul 1 2006

Keywords

  • Adsorption isotherm
  • Cu(II)-IDA
  • IMAC
  • Metal chelate affinity
  • RNA purification
  • Water elution

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Computer Vision and Pattern Recognition
  • Immunology
  • Molecular Biology

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