Isolation and purification of oligonucleotides by ion-exchange: Studies on the mechanism of separation

The retention behavior of selected synthetic oligonucleotides on a continuous-bed-matrix, non-porous, strong anion-exchange stationary phase UNO(TM) column from Bio-Rad has been investigated. Columns of this type have been used successfully in protein separations. The protein results are quite promising with advantages such as high recovery of biological activity, operation at low back-pressures, minimal nonspecific binding effects, and stationary phase stability over the pH range 2 to 12. These advantages are combined with the speed, resolution, and capacity provided by continuous bed technology. This dissertation reports the first successful use of this column type to resolve closely related oligonucleotides and focuses on experiments to clarify the retention mechanism.; Synthetic oligonucleotides that had been purified by PAGE (Polyacrylamide Gel Electrophoresis) and claimed to be highly pure have been purified further by gradient elution using the continuous bed, ion-exchange column. Separation of the N and N-1 oligomer has been achieved using a shallow mobile phase gradient. The temperature effect has been studied and retention increased linearly with increasing column temperature from -30°C to 50°C. Volatile buffer systems---e.g. triethylamine acetate---could be employed to purify oligonucleotides and no desalting procedure is required after the column separation step. The recoveries from the columns are 70% or higher. The maximum loading capacity of an analytical column UNO(TM) Q1 (7 mm ID x 35 mm length) was determined to be to be 14mg of 40mer oligomer.; The separation mechanism is predominantly anion-exchange but hydrophobic interaction plays an important role. Addition of organic solvent to the mobile phase modifiers affects the retention of oligomers significantly. Ion-exchange separations of pearl-chain oligonucleotides of the same length give different retention times. The retention sequence is A < T < C < G and A has the shortest retention time. Sequence order comparison has also been made with reversed-phase chromatography using C-4 column and with capillary gel electrophoresis separation. Those two other separation techniques give a retention (or for CE, migration) order of C < A < T < G, where C has the shortest retention (migration) time. Due to oligomer G's tendency to fold back and to form aggregates, an anomalously broad peak or even two broad peaks are shown with 20mer G HPLC experiments. Under denatured condition in CE experiments, only single peak is shown. CD results also support the formation of secondary structure is formed.; Dextran sulfate has been identified as a good candidate for the studies of the possible full development of a displacement train in columns such as the UNO Q1 (7mm x 35mm) thus testing an earlier report.