Significance of Nucleic Acid Melting Temperature in Liquid Chromatography Analysis was presented by Martin Gilar from Waters Corporation, USA.
The ability of nucleic acids to form duplex structures plays a central role in their analysis, purification, and enzymatic modification, according to Gilar. For example, small interfering RNA (siRNA) can be analyzed either as a duplex under non-denaturing conditions or as individual strands under denaturing LC. Guide RNA (gRNA) can adopt secondary structures necessary for CRISPR-Cas9 binding, which may influence its chromatographic behavior. In mRNA purification, hybridization between the poly(A) tail and an immobilized oligo-dT is commonly employed, while complementary strand binding can protect RNA from single-strand-specific nucleases, according to Gilar.
The effectiveness of these approaches depends on the stability of the nucleic acid duplex, which is defined by the melting temperature (Tm). In his presentation, Gilar explained how Tm is influenced by variables such as buffer concentration, ion type, organic solvent content, and the choice of stationary phase in LC. Using chemically modified siRNA as a model system, Gilar demonstrated how these factors affect duplex stability and, consequently, the outcome of chromatographic separations.
Szabolcs Fekete from Waters Corporation in Geneva, Switzerland, presented a talk entitled Enhancing Oligonucleotide RPLC Selectivity with Weak-to-Strong Ion Pairing Gradients.
Fekete presented a new approach to improve ion-pair reversed-phase (IP-RP) separations of oligonucleotides by using a dual ion-pairing gradient. While most methods rely on a fixed concentration of a single ion-pairing agent with an organic solvent gradient, Fekete proposed combining a weak, hydrophilic ion-pairing reagent in the initial aqueous phase with a strong, hydrophobic one in the later organic-rich phase. This method significantly improves resolution and selectivity, particularly for complex mixtures containing both size and sequence variants, according to Fekete. The technique, enhanced with concave gradients and short columns (20 x 2.1 mm), enables faster, high-resolution separations, supporting efficient analysis in oligonucleotide drug development. Fekete also described the benefits of dual and ternary gradient strategies for method development in oligo analysis.
Challenges in the Analytical and Preparative Separation of Therapeutic Oligonucleotides was presented by Torgny Fornstedt from Karlstad University, Sweden.
Fornstedt addressed the analytical challenges of separating complex therapeutic oligonucleotides, which often feature extensive chemical modifications and generate hundreds of impurities and diastereomers—especially in phosphorothioate-modified sequences. For a fully modified 20-mer, this can exceed half a million stereoisomers. Fornstedt emphasized ion-pair reversed-phase liquid chromatography (IP-RPLC) as the most effective method for both analytical and preparative separations. He highlighted the importance of selecting appropriate stationary phases, mobile phases, and gradient conditions. The presentation also introduced a digital, automated approach to resolve overlapping chromatographic peaks, allowing more accurate quantification from complex samples. Comparisons were made between antisense oligonucleotides (ASOs) and siRNA separation strategies.
