Purification of RNA Oligonucleotides using HPLC

Purification of RNA Oligonucleotides

Oligonucleotides are synthesized on solid phase supports using an automated procedure of sequential addition of one nucleotide residue. At the end of the synthesis the oligonucleotide remains attached to the support matrix with the nitrogen and oxygen protecting groups left intact. The cleavage from the solid support matrix removes the protecting groups with the exception of the dimethoxytrityl (DMT) protecting group attached to the 5'-O-oligonucleotide terminus. This DMT protecting group can be used to separate based on hydrophobicity and then following deprotection, removal of the DMT group, anion‑exchange HPLC can be performed to further resolve the oligonucleotide product from failure sequences.

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What is Synthetic oligonucleotides?

Synthetic oligonucleotides are promising therapeutic agents for the treatment of many diseases, including viral infections and cancer. Several classes of nucleic acids, such as antisense oligonucleotides, small interfering RNAs (siRNAs), and aptamers, are being investigated for therapeutic applications.

They are synthesized in a multistep process. Separation of the full-length product from the shorter failure sequences is a significant problem. This purification step becomes more difficult as the oligonucleotide length increases.

This application note demonstrates the purification workflow of a single-stranded RNA ON by ion-pair reversed-phase HPLC. A method is developed at analytical scale and then transferred to preparative conditions.