HPLC Purification of Long Synthetic Oligonucleotides

This application note demonstrates HPLC purification of long synthetic oligonucleotides.

HPLC Purification of Oligonucleotides

Synthetic oligonucleotides are utilized in a variety of molecular biology applications including polymerase chain reaction (PCR), DNA sequencing, and as antisense drugs. Purification of these oligonucleotides by traditional methods such as polyacrylamide gel electrophoresis (PAGE), can be time consuming, expensive, and more difficult on a larger scale. In addition, the synthesis of oligonucleotides greater than 50 bases in length is challenging, which results in a final product containing many failure impurities (<50% purity). A method has been developed for the reversed-phase high performance liquid chromatography (RP-HPLC) purification of long-chain synthetic oligonucleotides using traditional ionpairing (IP) mobile phases. Using a 4.6 × 50 mm analytical column, up to 0.25 μmole of synthetic DMToff oligonucleotide can be purified in one injection using ultraviolet (UV) detection. The method requires no post-purification desalting or detritylation.

Oligonucleotide Separation on XTerra MS C₁₈ Columns

XTerra Columns are packed with porous hybrid sorbent with particle sizes as low as 2.5 μm. The sorbent has high mass load capacity needed for semi-preparative and preparative purifications of long-chain synthetic oligonucleotides. The small sorbent particle diameter permits resolution of the target oligonucleotide from closely-eluting impurities (similar retention), even for oligonucleotides greater than 50mer in length. Figure 1 shows analytical scale RP-HPLC separation of a crude synthetic 60mer oligonucleotide under ion-pairing (IP) conditions. Many failure products can be observed in the chromatogram, however, they are all separated from the target product in under 20 minutes. Purity was determined by UV peak area at 260 nm.

Semi-Preparative Purification of Long Oligonucleotides

Figure 2 shows the analytical injection and subsequent purification of a synthetic 59mer oligonucleotide on the 50 nmole scale. The indicated collection window ensures maximum purity of the target fraction ( >80%).

Oligonucleotides synthesized on a 50 nmole–0.25 μmole scale can be purified using a 4.6 × 50 mm XTerra Column. However, it is recommended that a larger column size (i.e. 10 x 50 mm) be used to routinely purify semi-preparative scales (>0.1 μmole). Figure 3 shows the HPLC purification of the same 59mer from Figure 2 on a 0.25 μmole scale. Although the PDA detector is saturated at 260 nm, adjusting the detection wavelength to 290 nm allows one to decide on the appropriate collection window.