Department of Chemistry, University of Texas San Antonio, San Antonio, TX 78149, United States.
Department of Chemistry, University of Texas San Antonio, San Antonio, TX 78149, United States.
J Chromatogr A. 2014 Apr 25;1339:73-9. doi: 10.1016/j.chroma.2014.02.074. Epub 2014 Mar 3.
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.
为了在温和的反应和偶联条件下开发新形式的 DNA 偶联，合成了含有 3' 核糖核苷酸的 DNA 寡核苷酸。在与毫摩尔过碘酸钠（NaIO4）反应时，核糖在 pH6.8 下被氧化为二醛。该反应在 30 分钟内完成，用毫摩尔亚硫酸钠（Na2S2O5）淬灭，然后适合与酰肼琼脂糖载体偶联。偶联的半衰期为 27 分钟，80%在 2 小时内偶联。与 CAAT 增强子结合蛋白（C/EBP）结合的 EP18 寡核苷酸用 3' 核糖（rEP18）合成并与酰肼琼脂糖偶联。制备的柱子在 50 天后没有明显的寡核苷酸损失。从表达 GFP-C/EBP 嵌合融合蛋白的细胞的粗细菌提取物应用于柱子，并在不同盐浓度下洗脱。活性蛋白在 0.5M NaCl 中洗脱，SDS-PAGE/银染色凝胶显示一条主要条带，与 GFP-C/EBP 以及三种次要污染物共迁移。这为使用温和化学将 DNA 偶联到固体载体提供了一种新的替代方法，这种方法对 DNA 没有损害，如果需要，也可以在核提取物存在的情况下进行。
