Optimizing purification of oligonucleotides with reversed phase trityl-on solid phase extraction

Abstract: Oligonucleotides are synthetic strings of DNA or RNA used mostly for biochemical analysis and diagnostics. For them to be useful in these fields, a purity over 90% is most often required. However, when synthesizing these sequences, many “failures” (shorter sequences) are made in the step-wise process. The synthesized oligonucleotides need to therefore be purified. This is most often done with gel electrophoresis or liquid chromatography. These methods are, on the other hand, very time-consuming and laborious. Solid phase extraction (SPE) is a much faster purification method if optimized and it can be done with the standard cartridges as well as 96-well plates, that allow many samples to efficiently be run at the same time. With reversed phase (RP) SPE, the dimethoxytrityl (DMT) group, that is attached to the target at the final synthesis step, can be used for stronger retention to the bed sorbent and leaving only the target at the final eluting stage. The impurities without a DMT-on group, that do not adsorb to the sorbent, are washed away in earlier steps. The purpose of this study is to optimize an SPE method for purification of oligonucleotides. Two different cartridges, Clarity QSP (Phenomenex) and Glen-Pak (Glen Research) were used. The purity analysis and oligonucleotide identification were done using anion exchange - high performance liquid chromatography (AIE-HPLC) and time-of-flight mass spectrometry (TOF MS). To conclude, Clarity QSP achieved, at the most, a purity of 68.8% with the recommended SPE steps by Phenomenex. Alterations in the extraction procedure resulted in similar purity or lower. Glen-Pak reached a peak purity of 78.8% when doing a double salt wash of 5% ACN in 2 M sodium chloride and another double wash after detritylation with 1% acetonitrile. This method has to be further optimized in order to reach a purity of at least 90% to be useful in industrial settings.