Improved Purification of sgRNA and pegRNA by Use of a 5'-Terminal Dioctyloxytrityl (DOT) Protecting Group.

The purity and chemical integrity of oligonucleotides are critical determinants of safety, efficacy, and reproducibility in RNA therapeutics. Impurities arising from incomplete synthesis, degradation, or side reactions can compromise pharmacological performance and induce off-target or immune responses. For long, structured oligonucleotides such as CRISPR guides, purification can be costly in both resources and yield. To overcome these limitations, we developed a purification method based on a 5'-terminal dioctyloxytrityl (DOT) protecting group. In this purification method, DOT-protected full-length compounds show high-resolution separation from truncated impurities. The DOT group can then be removed under mild, rapid, and scalable conditions. DOT chemistry is analogous to "DMT-ON" approaches that are well-known and widely used in the field, and is fully compatible with existing synthesis workflows, but dramatically improves HPLC purification relative to DMT-ON approaches by providing higher retention and thus better resolution to enable the purification of long, complex, and chemically modified sequences.