Enhanced Oligonucleotide Purity
Interestingly, the purity of the oligonucleotides obtained using our cartridge model system was better than that provided by standard cartridge purification. This prompted us to extend our investigation and delve deeper into our data, allowing us to explore whether tocopherol conjugation could be used as an efficient method of oligonucleotide purification.
Examples of the IE-HPLC analysis of the resultant oligonucleotides are shown in Figure 2. Specifically, expansion around the main peak illustrates synthesis failures. Although the standard purification method gives a relatively clean product when compared with the crude oligonucleotide, the tocopherol-based method clearly shows fewer failures.
In order to demonstrate the functionality of the pure oligonucleotide, conjugation of thiol-labeled oligonucleotides (prepared with and without tocopherol) was carried out using 6-iodofluorescein (6-IAF), which is a common dye used in many detection applications. For example, this is often used to incorporate a fluorescein moiety into oligonucleotides used for detection.
We have found that as the purity of the thiol-modified oligonucleotide increases, the efficiency of conjugation also increases. Use of the crude oligonucleotide gave ~80% labeling, whereas standard cartridge purification gave ~86% labeling and Link Technologies’ tocopherol/cartridge method gave >90% labeling (Figure 3).
This clearly shows that the use of oligonucleotides purified using tocopherol results in a more efficient conjugation than either standard cartridge purification or using the oligonucleotide in its crude form. We have also demonstrated that the specific activity of the thiol-modified oligonucleotide is not adversely affected by the use of tocopherol.
While the method has been described for the purification of thiol-modified oligonucleotides, this can also be applied to other modified oligonucleotides in those cases where it is possible to use a cleavable linker.