A robust and versatile enzyme enabling seamless integration into RNA synthesis protocols
Double-stranded RNA (dsRNA) is a significant contaminant in mRNA. During in vitro transcription (IVT), RNA polymerases naturally generate dsRNA by products through a number of mechanisms including cryptic promoters or loopback RNAs. Once inside cells or animals, these dsRNA contaminants can trigger innate immune activation and diminish protein expression, thereby jeopardizing the safety, tolerability, and efficacy of the potential therapeutic product.
Recently, novel mutant enzymes have been developed to replace the commonly used wild-type T7 RNA polymerase to minimize dsRNA formation in IVT. While a theoretically simple approach, some enzymes may necessitate IVT condition optimization — especially when used in co-transcriptional capping — to avoid adverse effects on mRNA quality attributes such as yield, integrity, and capping efficiency.
In this tech note, we report a novel RNA polymerase that can:
Reduce dsRNA formation across various constructs, CleanCap cap analogs, and modified nucleotides
Directly replace wild-type T7 RNA polymerase in diverse IVT protocols without compromising other critical quality attributes
Efficiently synthesize long and complex templates like saRNAs while minimizing dsRNA formation
Improve mRNA performance by lowering innate inflammatory responses and increasing protein expression
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