PEGylated Graphene Oxide Enhances In Vitro Transcription by Suppressing RNA Rebinding to T7 Polymerase.

In vitro transcription (IVT) using T7 RNA polymerase is a key step in mRNA synthesis for therapeutic applications. However, the generation of double-stranded RNA (dsRNA) byproducts during IVT─primarily due to RNA rebinding and self-priming─triggers innate immune responses and reduces translation efficiency. Here, we present a simple and effective strategy to minimize dsRNA formation during IVT by incorporating PEGylated graphene oxide (PEG-GO). Graphene oxide (GO) preferentially binds single-stranded nucleic acids, but its use is limited by protein adsorption and low solubility in Mg2+-containing buffers. PEG modification improves GO's dispersibility and reduces protein binding, allowing selective sequestration of nascent RNA without inhibiting T7 RNA polymerase activity. The addition of PEG-GO to the IVT reaction reduced the dsRNA content by over 75% while maintaining RNA yield and accelerating transcription kinetics. Moreover, mRNA synthesized in the presence of PEG-GO showed enhanced protein expression and reduced interferon-β secretion in transfected cells, comparable to post-IVT-purified mRNA. Our work demonstrates PEG-GO as a practical additive for improving the quality and scalability of IVT-based mRNA production.