AI Insight
Researchers engineered a series of chimeric T7 RNA polymerase (T7 RNAP) variants by fusing optimized polymerase mutants with DNA-binding domains such as Sso7d and MC1, creating enzymes with improved salt tolerance, catalytic activity, and template selectivity. These chimeric mutants functioned effectively at NaCl concentrations up to 270 mM and reduced the formation of immunogenic double-stranded RNA (dsRNA) byproducts to below 0.001%, while also improving transcript integrity and 3' end homogeneity. When applied to circular RNA synthesis, the lead variants enabled a simplified non-fed-batch process with high initial nucleotide concentrations, achieving RNA titers of 15 mg/mL, representing a 50% yield increase over standard approaches.
Why it matters
Improving the purity and yield of in vitro transcription is directly relevant to the manufacturing of mRNA and circular RNA therapeutics, where dsRNA contamination is a known trigger of unwanted immune responses that can reduce therapeutic safety and efficacy. These engineered enzymes could simplify industrial-scale RNA production workflows while simultaneously improving product quality for clinical applications such as mRNA vaccines and gene therapies.
⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
The rapid advancement of mRNA therapeutics has imposed stringent requirements on both the quality and scalability of in vitro transcription (IVT) products. However, the accumulation of double-stranded RNA (dsRNA) byproducts and 3′-terminal heterogeneity during T7 RNA polymerase (T7 RNAP)-mediated transcription can robustly trigger deleterious innate immune responses and compromise translation efficiency. Existing enzyme engineering strategies frequently struggle to reconcile the trade-offs between salt tolerance, volumetric productivity, and product purity. Here, we report a novel engineering strategy for salt-tolerant T7 RNAP by fusing optimized mutant polymerases with diverse DNA-binding domains (e.g., Sso7d, MC1). This approach orchestrated the development of a series of chimeric T7 RNAP mutants designed to bolster catalytic activity and template selectivity under high-ionic-strength conditions while concurrently suppressing RNA-dependent RNA polymerase (RdRP) activity. Our lead chimeric mutants exhibited exceptional salt tolerance and processivity in the presence of up to 270 mM NaCl. Notably, these mutants significantly diminished dsRNA formation to less than 0.001%, while markedly improving transcript integrity and 3′ homogeneity, thereby facilitating superior translation efficiency for both linear mRNA and circular RNA (circRNA). Crucially, this heightened salt tolerance does not necessitate a trade-off in RNA yield, affording broader flexibility for downstream process optimization. In an enzymatic circRNA synthesis system, these mutants enabled a non-fed-batch configuration with high initial rNTP concentrations (15 mM each), resulting in a 50% increase in yield and achieving an unprecedented titer of 15 mg/mL. This research provides a robust enzymological solution that harmonizes quality and productivity for the industrial-scale manufacturing of high-concentration, low-immunogenicity RNA.