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Double-stranded RNA (dsRNA) formation during in vitro transcription is a significant challenge due to its potent immunogenic properties. In this chapter, we present an optimized chromatographic method for purifying linearized DNA templates, effectively reducing dsRNA impurities during mRNA production. The findings highlight the critical role of template quality, demonstrating how template impurities directly contribute to dsRNA formation. Moreover, we show that additional polishing purification steps further decrease dsRNA levels, reducing the activation of innate immune responses mediated by dsRNA-sensing receptors such as Toll-like receptor 3.

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Hydrophobic Chromatography Purification of Linearized Plasmid DNA Reduces dsRNA Formation During In Vitro Transcription

  • 实验方案

  • 发表日期：

    2025年

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  • Juan Martínez-Oliván,
  • Verónica Lampaya,
  • Ana Larraga,
  • Héctor Magallón,
  • Julia Domínguez,
  • Víctor Navarro,
  • Diego Casabona

摘要

Double-stranded RNA (dsRNA) formation during in vitro transcription is a significant challenge due to its potent immunogenic properties. In this chapter, we present an optimized chromatographic method for purifying linearized DNA templates, effectively reducing dsRNA impurities during mRNA production. The findings highlight the critical role of template quality, demonstrating how template impurities directly contribute to dsRNA formation. Moreover, we show that additional polishing purification steps further decrease dsRNA levels, reducing the activation of innate immune responses mediated by dsRNA-sensing receptors such as Toll-like receptor 3.