DNA Nanoflowers Assemble via Template-Induced Crystallization during mRNA Synthesis

mRNA-based vaccines and therapeutics are synthesized using the in vitro transcription (IVT) reaction. However, the byproduct pyrophosphate (PPi) forms insoluble magnesium pyrophosphate (Mg 2 PPi) during IVT, which coprecipitates with DNA, the transcription blueprint, interrupting the reaction and reducing RNA yield. The severity of this crystallization problem has prompted the routine inclusion of inorganic pyrophosphatase (PPase) in industrial processes to hydrolyze pyrophosphate into phosphate ions and prevent crystallization. To date, the mechanism by which Mg 2 PPi crystals interact with DNA has remained unclear. In this work, we demonstrate that DNA promotes Mg 2 PPi crystallization through a template-directed mechanism, reducing the nucleation induction time by a factor of 2. The resulting crystals form compact spherulites with a rosette habit, distinct from the morphologies observed in DNA-free systems. We propose that DNA preferentially templates the nucleation of Mg 2 PPi·3.5H 2 O crystals by interacting with the {100} crystal face. These findings reveal how DNA influences Mg 2 PPi crystallization and offer mechanistic insights relevant to optimizing RNA manufacturing systems.