Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement.

In vitro transcription (IVT) using T7 RNA polymerase is a cornerstone technology for synthetic messenger RNA (mRNA) production. However, a persistent challenge is the formation of immunogenic double-stranded RNA (dsRNA) byproducts, primarily arising from promoter-independent antisense transcription at free DNA ends. Here, we introduce the nicked low dsRNA template (NiLoT) strategy, a template engineering approach in which a single-strand nick is placed in the non-template DNA strand to promote R-loop formation and suppress antisense RNA synthesis. NiLoT significantly reduces dsRNA contamination without compromising RNA yield and enhances translational output while minimizing innate immune activation in human cells. This broadly applicable method improves IVT mRNA quality and supports more efficient and scalable RNA production.