Ion-pairing reversed-phase liquid chromatography (IP-RPLC), a standard method to characterize small oligonucleotides, can also separate large RNA molecules. Its advantages extend beyond simple separation by size and sequence.
Scientists at the University of Geneva report that IP-RPLC avoids many challenges typically associated with anion exchange chromatography or ultra-wide pore size-exclusion chromatography columns, while also being compatible with mass spectrometry. As yet, however, not much is known about the effects of various ion-pairing agents (IPAs) on large RNA separation.
To help bridge that knowledge gap, analytical chemist Jonathan Maurer, PhD, a postdoctoral researcher, and colleagues at the University of Geneva in Switzerland, assessed 13 IAPs of varying hydrophobicity levels. They aimed to determine the agents and conditions that were most effective at improving RNA retention and selectivity. Agents were considered alone and in combination, at different concentrations, between 45° C and 85° C, and in pH ranges from 6 to 8.
Best resolution
“Combining a weakly hydrophobic IPA with a moderately hydrophobic one further improved resolution, by taking advantage of the complementary properties of the IPAs,” Maurer’s team reported in a recent paper.
For RNA species up to 6,000 nucleotides, they obtained the best resolution by combining 100 mM of butylammonium acetate (BA) and 50 mM tripropylammonium acetate (TPA) as the IPAs. This enabled separation of multiple RNAs of similar sizes and improved resolution by 35%.
“The downside was that the supermacroporous polymeric (divinylbenzene) column used did not efficiently retain the nucleoside triphosphates,” Maurer said. This was problematic because these molecules are used as starting material for in vitro transcription RNA production.
“Adding a more retentive post-column composed of a more classical C18 stationary phase resolved that issue. The developed method efficiently separates nucleic acids ranging from 1 to 6,000 nucleotides,” Maurer added.
In terms of separation, the best results were achieved with a pH of 7.0 and a 65°C column temperature while using acetonitrile as the organic solvent, and by combining TPA and BA as ion-pairing agents.
Conversely, most other published reports used triethylammonium acetate (TEA) as the ion-pairing agent. TEA’s low level of hydrophobicity, however, enabled only poor resolution, especially once RNA exceeded 1,500 nucleotides. “Separation improved as IPA hydrophobicity increased,” the scientists reported.
“Highly hydrophobic IPAs, however, come with other issues, such as poor solubility, high adsorption behavior, and high toxicity for humans and environment,” Maurer pointed out. “Because they must be used at lower concentrations (25 mM), this method significantly reduced their separation power.” Therefore, “only the moderately hydrophobic IPAs provided effective separation across the ladder.
“By describing the best conditions to analyze large RNAs by IP-RPLC,” Maurer said, “this paves the way for the routine use of this chromatography technique to evaluate RNA integrity. This is a key critical quality attribute of newly developed mRNA-based therapeutics.”
