A METHOD FOR THE PURIFICATION OF OLIGONUCLEOTIDES CONTAINING STRONG INTRAMOLECULAR OR INTERMOLECULAR INTERACTIONS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

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 hav e 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 ty pe of oligonucleotide can be purified on an ion-exchange column by add ing formamide to the mobile phase, the resulting resolution is poor an d the formamide must subsequently be removed from the purified product . We find that simply having 20% formamide in the loading buffer is su fficient 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 3 5 ODs have been satisfactorily purified with recoveries of 95% or bett er. This procedure was particularly suitable for purification of oligo nucleotides containing base-labile modifications, such as acetylaminof luorene-modified oligonucleotides, since other denaturing HPLC purific ation methods usually employ strong alkaline conditions or high temper atures that might result in damage to the adduct. (C) 1995 Academic Pr ess, Inc.