A METHOD FOR THE PURIFICATION OF OLIGONUCLEOTIDES CONTAINING STRONG INTRAMOLECULAR OR INTERMOLECULAR INTERACTIONS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
A METHOD FOR THE PURIFICATION OF OLIGONUCLEOTIDES CONTAINING STRONG INTRAMOLECULAR OR INTERMOLECULAR INTERACTIONS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
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DOI：
10.1006/abio.1995.1521
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发表时间：
1995-10-10
影响因子：
2.9
通讯作者：
ROMANO, LJ
中科院分区：
文献类型：
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作者：
ARGHAVANI, MB;ROMANO, LJ
含有高鸟嘌呤含量的合成寡脱氧核糖核苷酸倾向于在溶液中形成分子内或分子间复合物，这使得HPLC纯化困难或有时不可能。我们已经开发了一种简单的方法，使我们能够纯化一系列的高度鸟嘌呤丰富和自我互补的寡核苷酸，通过HPLC反相PRP-1柱。尽管其他人已经表明，这种类型的寡核苷酸可以通过向移动的相中加入甲酰胺而在离子交换柱上纯化，但所得的分离度很差，并且随后必须从纯化的产物中除去甲酰胺。我们发现，简单地在上样缓冲液中含有20%甲酰胺就足以去除干扰相互作用。少量甲酰胺快速通过反相色谱柱，远离寡核苷酸的峰位置。高达35个ODs已令人满意地纯化，回收率为95%或更好。该方法特别适用于纯化含有碱基不稳定修饰的寡核苷酸，如乙酰氨基芴修饰的寡核苷酸，因为其他变性HPLC纯化方法通常采用可能导致加合物损坏的强碱条件或高温。(C)出版社：Academic Press
Synthetic oligodeoxyribonucleotides containing a high guanine content have a tendency to form intra- or intermolecular complexes in solution that make HPLC purification difficult or sometimes impossible. We have developed a simple method that has enabled us to purify a series of highly guanine-rich and self-complementary oligonucleotides by HPLC on a reverse-phase PRP-1 column. Although others have shown that this type of oligonucleotide can be purified on an ion-exchange column by adding formamide to the mobile phase, the resulting resolution is poor and the formamide must subsequently be removed from the purified product. We find that simply having 20% formamide in the loading buffer is sufficient to remove the interfering interactions. This small amount of formamide passes quickly through the reverse-phase column, far removed from the peak position of the oligonucleotides. Quantities of up to 35 ODs have been satisfactorily purified with recoveries of 95% or better. This procedure was particularly suitable for purification of oligonucleotides containing base-labile modifications, such as acetylaminofluorene-modified oligonucleotides, since other denaturing HPLC purification methods usually employ strong alkaline conditions or high temperatures that might result in damage to the adduct. (C) 1995 Academic Press, Inc.