Nucleic acids researchJan 15, 2026

Template nicking reduces unwanted opposite-strand RNA production during in vitro transcription by displacing strands through R-loop formation

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jun 27, 2026

Abstract

Essence

A nicked DNA template strategy reduced immunogenic dsRNA byproducts while preserving IVT mRNA yield.

Evidence

Template-engineering experiments in T7 IVT and human cell assays tested NiLoT effects on dsRNA contamination, RNA yield, translation, and innate immune activation.

Caveat

The results are platform and cell-assay outcomes, so therapeutic performance and in vivo behavior remain untested in the abstract.

Simplified

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.

Full Text

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Funding

Competing interests

20%of authors

1 of 5

authors report competing interests

4 report none

PubMed

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