title: 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.
authors.affiliation: Department of Chemistry, University of Texas San Antonio, San Antonio, TX 78149, United States.
authors.forename: Yinshan
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Department of Chemistry, University of Texas San Antonio, San Antonio, TX 78149, United States.
authors.forename: Oleg
authors.initials: O
authors.lastname: Larionov
authors.affiliation: Department of Chemistry, University of Texas San Antonio, San Antonio, TX 78149, United States. Electronic address: harry.jarrett@utsa.edu.
authors.forename: Harry W
authors.initials: HW
authors.lastname: Jarrett
mesh_terms: D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D002846:Chromatography, Affinity; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009843:Oligoribonucleotides / Q000737:chemistry*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*; D012266:Ribose / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D009843:Oligoribonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D012266:Ribose; D004247:DNA; D012685:Sepharose
keywords: Affinity chromatography; Chemical coupling; DNA; Transcription factors