Coupling of deoxyribonucleic acid to solid supports using 3' terminal ribose incorporation

Abstract

To develop a new form of DNA coupling under mild reaction and coupling conditions, DNA oligonucleotides were synthesized containing a 3' ribonucleotide. Upon reaction with millimolar sodium metaperiodate (NaIO4), the ribose is oxidized to a dialdehyde at pH 6.8. This reaction is complete in 30min, is quenched with millimolar sodium metabisulfite (Na2S2O5) and is then suitable for coupling to hydrazide-agarose supports. Coupling occurs with a half-time of 27min and 80% couples in 2h. The EP18 oligonucleotide which binds to the CAAT enhancer binding protein (C/EBP) was synthesized with a 3' ribose (rEP18) and coupled to hydrazide-agarose. The columns prepared show no significant loss of the oligonucleotide after 50 days. A crude bacterial extract from cells expressing a chimeric fusion protein of GFP-C/EBP was applied to the columns and eluted with different salt concentrations. The active protein elutes in 0.5M NaCl and SDS-PAGE/silver stained gels show a single major band which comigrates with GFP-C/EBP as well as three minor contaminants. This provides a new alternative way of coupling DNA to solid supports using mild chemistry which is non-detrimental to the DNA and can be performed if required in the presence of nuclear extract.

Keywords: Affinity chromatography; Chemical coupling; DNA; Transcription factors.