High-Resolution Separation of Oligonucleotides on a Pellicular Anion-Exchange Column

Multiple oligonucleotide (ON)-based therapeutic models have been developed to date including antisense oligonucleotides (ASOs), aptamer oligonucleotides (AOs) and short interfering RNA oligonucleotides (siRNA). In addition, modifications to oligonucleotide base-, backbone-, and ribosemoieties have all been introduced to limit nuclease-mediated degradation in therapeutic ONs. Mechanisms to resolve oligonucleotides from their modified forms, synthetic failure fragments, and their metabolic products are therefore necessary, although these can be technically challenging to develop. The Thermo ScientificTM DNAPacTM PA200 column offers industryleading selectivity and resolution for the separation of therapeutic and diagnostic oligonucleotides. demonstrated oligonucleotide purity improves applications to regulatory agencies. Oligonucleotide characteristics influencing chromatographic interactions include length, base composition, the presence of coupling failures, and dyes (or ‘‘molecular beacons’’); in addition residual-protecting groups, employed during singlestranded nucleic acid (ssNA) synthesis, can also have an effect. Each modification may be combined with the others in a given oligonucleotide. Oligonucleotide assays are typically accomplished by anion-exchange or ion-pair reversed-phase chromatography. Both approaches employ ionic interactions between the analytes, either directly with the stationary phase (i.e. anion exchange) or with the ion-pair reagent, which in turn interacts with the stationary phase. Both approaches enjoy wide popularity as both often deliver good resolution and analysis throughput for