Size-exclusion chromatography with post-column nucleic acid staining and fluorescence detection for sensitive ribonucleic acid analysis

RNA-based therapeutics, such as vaccines or gene therapy applications, are steadily gaining in popularity and require extensive analytical characterization with several orthogonal methods to ensure highest purity for safe use. In this study, size-exclusion chromatography (SEC) with multiple detectors was used for characterizing single- and double-stranded (ss/ds) RNA ladders, providing insights into how conformation and viscosity affect the different elution profiles observed for both species in SEC. Furthermore, high-resolution chromatographic separation was merged with the high-sensitivity detection provided by fluorescent dyes by developing an innovative approach relying on SEC with post-column nucleic acid staining and fluorescence detection (SEC-pcs-FLD). Herein, SEC-pcs-FLD was successfully applied to four out of five investigated dyes, namely SYBR Gold, SYBR Green I, Thiazole Orange, and YOYO-1, yielding a 10- to 150-fold sensitivity increase for mRNA detection compared to typically applied UV detection for chromatographic nucleic acid analysis. Besides the significant sensitivity increase, reproducibility and linearity regarding the response to concentration ratio were demonstrated for the post-column staining approach, indicating suitability for low-level RNA quantitation. Two of the investigated dyes, namely SYBR Green I and YOYO-1, further allowed for the differentiation of ssRNA and dsRNA species based on SEC-pcs-FLD, as they showed stronger affinity towards dsRNA reflected in a roughly five- to sixfold higher FLD/UV response ratio compared to ssRNA. This approach consequently also holds promise for the quantitation of dsRNA impurities potentially formed during mRNA synthesis and associated with causing innate immune responses, especially as currently applied methods like immunoassays show several limitations.