A method for the purification of oligonucleotides containing strong intra- 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 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 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.