A METHOD FOR THE PURIFICATION OF OLIGONUCLEOTIDES CONTAINING STRONG INTRAMOLECULAR OR INTERMOLECULAR INTERACTIONS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY
Mb. Arghavani et Lj. Romano, A METHOD FOR THE PURIFICATION OF OLIGONUCLEOTIDES CONTAINING STRONG INTRAMOLECULAR OR INTERMOLECULAR INTERACTIONS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY, Analytical biochemistry, 231(1), 1995, pp. 201-209
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.