The in vitro transcription (IVT) reaction, utilized for the synthesis of mRNA, also generates immune-stimulating double-stranded RNA contaminants. Traditional methods to address this issue, such as reversed-phase chromatography, necessitate the use of organic solvents, which may pose environmental and safety concerns. In this application note, we introduce an innovative approach to dsRNA removal utilizing classic affinity chromatography.
Following IVT and initial capture step on CIMmultus Oligo dT18, the mRNA with dsRNA impurities undergoes a brief incubation in a buffer with acidic pH (below 4), effectively denaturing the hydrogen bonds within dsRNA. This process is followed by a re-loading step onto the CIMmultus Oligo dT18 column, ensuring the purification of polyadenylated mRNA devoid of dsRNA contaminants. Our approach provides a solvent-free, efficient, and environmentally friendly solution for dsRNA removal, enhancing the purity and safety of mRNA products, all while using the tried-and-true affinity monolith, the CIMmultus Oligo dT18.
