Studying Mobile and Stationary Phases’ Effects on siRNA Stability

Scientists from Waters Corporation in Milford, Massachusetts recently published a study in the Journal of Chromatography A, examining how different mobile and stationary phases affect small interfering RNA (siRNA) duplex stability in liquid chromatography (1).

Ribosome as part of an biological cell constructing messenger rna molecules - 3d illustration | Image Credit: © Christoph Burgstedt - stock.adobe.com

In this study, the scientists investigated the impact of mobile phase composition on the melting temperatures of siRNA duplexes. siRNAs are short, double-stranded RNAs that dissociate to single strands and bind to specifically target messenger RNA (mRNA) sequences (2). They explored mobile phases commonly used in ion-pair reverse-phase-LC (IP-RP-LC), hydrophilic interaction chromatography (HILIC), ion-exchange LC and size-exclusion chromatography (SEC).

Oligonucleotide analysis in liquid chromatography (LC) is typically performed under denaturing conditions using an elevated temperature. High-column temperatures ensure that oligonucleotides that tend to form intra- (hairpin loops) and inter-molecular (G-quadruplexes) interactions are denatured and have predictable retention behaviors. Further, melting temperature (T_m) is the property used to measure duplex stability. Tm is the temperature at which half of the double stranded duplex is dissociated into the complementary strands or alternatively when half of the secondary structure (intramolecular hairpin) is linearized. However, it is unknown how chromatographic conditions and mobile phase composition affect the duplex stability. High T_m values indicate that nucleic acids can form stable duplexes or secondary (self-complementary) structures. This occurs with nucleic acids with high cytidine and guanosine content or for modified nucleic acids, such as locked nucleic acids or 2′-modified oligonucleotides.

The researchers investigated siRNA melting temperature using differential scanning calorimetry (DSC), a thermal analysis apparatus measuring how physical properties of a sample change, along with temperature against time; this was used for solutions containing alkali metal cations, magnesium, ammonia, triethylamine or hexylamine (3). For selected mobile phases, the effects of adding organic solvents (methanol, acetonitrile, ethanol, and hexafluoroisopropanol) were also investigated. In the second part of the study, the team measured Tm of siRNA samples with the aforementioned chromatographic methods using different experimental column temperatures in hopes of observing on-column duplex melting.

They explored 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_m was 67.1–78.2 °C for 10–100 mM [Na+] concentration). It was found that the melting temperature decreased with increases in cation sizes. Including 20% of the organic solvent in buffer reduced the melting temperature by 1–3 °C, with denaturation power increasing in the order MeOH < EtOH < MeCN. Meanwhile, adding 100 mM hexafluoroisopropanol (HFIP) lowered the observed T_m by 1–2 °C.

The scientists also monitored siRNA melting temperatures using the chromatographic techniques within the 10–90 °C column temperature range. With SEC in 25 mM sodium phosphate buffer, the T_m was approximately 70 °C, about 5 °C lower compared to average DSC values. Further, since in SEC separation mode, neither the analytes nor the mobile phases interacted with the stationary phase, the observed T_m agreed with DSC results. Meanwhile, HILIC yielded significantly higher T_m and RP-LC significantly lower T_m than expected. With these findings, it was concluded that an aqueous layer in HILIC or acetonitrile-rich layer in RP LC is a cause of the apparent differences in the experimental T_m. IP-RP-LC provided higher T_m compared to RP LC, in spite of the former method having a higher acetonitrile concentration. This layer is probably disrupted by the adsorption of charged ion-pairing reagents on the C18 surface.

References

(1) Gilar, M.; Redstone, S.; Gomes, A. Impact of Mobile and Stationary Phases on siRNA Duplex Stability in Liquid Chromatography. J. Chromatogr. A 2024, 1733, 465285. DOI: 10.1016/j.chroma.2024.465285

(2) Small Interfering RNA. Elsevier B.V. 2024. https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/small-interfering-rna (accessed 2024-8-27)

(3) Gill, P.; Moghadam, T. T.; Ranjbar, B. Differential Scanning Calorimetry Techniques: Applications in Biology and Nanoscience. J. Biomol. Tech. 2010, 21 (4), 167–193. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977967/