Impact of mobile and stationary phases on siRNA duplex stability in liquid chromatography.

Nucleic acid duplexes are typically analyzed in non-denaturing conditions. Melting temperature (T) is the property used to measure duplex stability; however, it is not known how the chromatographic conditions and mobile phase composition affect the duplex stability. We employed differential scanning calorimetry (DSC) method to measure the melting temperature of chemically modified silencing RNA duplex (21 base pairs, 0.15 mM duplex concentration) in mobile phases commonly used in reversed-phase, ion-pair reversed-phase, size exclusion and hydrophilic interaction chromatography. We investigated mobile phases consisting of ammonium acetate, alkylammonium ion-pairing reagents, alkali-ion chlorides, magnesium chloride, and additives including methanol, ethanol, acetonitrile and hexafluoroisopropanol. Increasing buffer concentration enhanced the duplex stability (T was 67.1 - 78.2 °C for 10-100 mM [Na] concentration). The melting temperature decreases with the increase in cation size (70.2 °C in 10 mM [Li], 68.1 °C in 10 mM [NH], 65.6 °C in 10 mM [Cs], and 56.6 °C in 10 mM [triethylammonium] solutions). Inclusion of 20 % of organic solvent in buffer reduced the melting temperature by 1-3 °C, and denaturation power increases in the order MeOH<EtOH<MeCN. Next, we investigated the RNA duplex melting using selected chromatographic techniques and within the 10-90 °C column temperature range. Melting temperature obtained with size exclusion chromatography in 25 mM sodium phosphate buffer was ∼ 70 °C, about 5 °C lower compared to DSC values. The apparent melting temperature for a reversed-phase chromatography experiment was < 10 °C for 10 mM ammonium acetate mobile phase with acetonitrile eluent. Ion-pair reversed-phase with 10 mM triethylamine, 100 mM hexafluoroisopropanol and MeCN as eluent yielded T 49.3-54.9 °C. Hydrophilic interaction chromatography experiments with 10 mM ammonium acetate and ∼ 50 % acetonitrile mobile phase showed high duplex stability (T ∼ 80 °C). The observed chromatographic T values suggest that the formation of an organic/aqueous solvent layer on the chromatographic sorbent surface affects the duplex stability more significantly than the composition of the mobile phase alone.