Analytical and functional clearance of dsRNA contaminants from in vitro transcribed mRNA by an engineered dsRNA-binding protein

Double-stranded RNA (dsRNA) is a biologically active contaminant of in vitro transcribed (IVT) mRNA that can reduce protein expression, induce cytokines, affect cell viability, and confound experimental results. Here, we show that dsRNA contaminants containing modified nucleotides are difficult to quantify and functionally assess. Common antibody-based dsRNA ELISAs under-detected pseudouridine- and N1-methylpseudouridine-modified dsRNA, while a higher-sensitivity ELISA improved detection but remained chemistry dependent. Defined dsRNA spike-in experiments showed that effects of dsRNA on cell viability, cytokine induction, and protein expression were chemistry dependent and occurred at low burdens of 0.05-0.5 ng dsRNA per ug RNA. Analytical dsRNA depletion across several workflows did not always predict suppression of cytokine induction, supporting the need for a functional cell-based readout. We engineered B2-S, a single-chain spliced variant of the Flock House virus B2 dsRNA-binding protein, to selectively deplete dsRNA from crude IVT reactions while preserving mRNA recovery and integrity. Among tested methods, B2-S was the only non-enzymatic, non-chromatographic workflow that reduced the cytokine marker IP-10/CXCL10 to mock-transfection levels. These findings establish a paired analytical and functional framework for IVT mRNA quality assessment and introduce B2-S as an accessible method for producing mRNA with improved potency and reduced immunostimulatory activity.