Coupling of deoxyribonucleic acid to solid supports using 3' terminalribose incorporation

摘要

To develop a new form of DNA coupling under mild reaction and coupling conditions, DNA oligonu-cleotides were synthesized containing a 3' ribonucleotide. Upon reaction with millimolar sodiummetaperiodate (NaIO_4), the ribose is oxidized to a dialdehyde at pH 6.8. This reaction is complete in30 min, is quenched with millimolar sodium metabisulfite (Na_2S_2O_5) and is then suitable for coupling tohydrazide-agarose supports. Coupling occurs with a half-time of 27 min and 80% couples in 2 h. The EP18oligonucleotide 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 theoligonucleotide after 50 days. A crude bacterial extract from cells expressing a chimeric fusion protein ofGFP-C/EBP was applied to the columns and eluted with different salt concentrations. The active proteinelutes in 0.5 M NaCl and SDS-PAGE/silver stained gels show a single major band which comigrates withGFP-C/EBP as well as three minor contaminants. This provides a new alternative way of coupling DNAto solid supports using mild chemistry which is non-detrimental to the DNA and can be performed ifrequired in the presence of nuclear extract.