Engineering T7 RNA Polymerase for High-Purity In Vitro Transcription
In vitro transcription using bacteriophage T7 RNA polymerase (T7 RNAP) is the gold-standard platform for RNA production in both research and therapeutic applications. Despite its high processivity and promoter specificity, T7 RNAP generates multiple RNA by-products, including double-stranded RNA, 3'-extended transcripts, abortive RNAs, and prematurely terminated products. These impurities reduce RNA yield, complicate downstream purification, and raise safety concerns for RNA-based therapeutics by activating adverse innate immune pathways. Although reaction optimization and downstream purification strategies can mitigate these issues, they typically involve trade-offs between RNA purity and yield. Enzyme engineering has therefore emerged as a powerful upstream strategy to suppress by-product formation at its molecular origin. Here, we synthesize current knowledge on the structural and mechanistic basis of T7 RNAP by-product formation and systematically review engineering strategies to improve RNA purity. T7 RNAP variants are classified according to their underlying mechanisms of action, including enhanced thermostability, reduced non-specific template binding, smoother initiation-to-elongation transition, reduced premature termination, and template-biased polymerase designs. This analysis identifies general principles governing the trade-off between specificity and processivity and highlights synergistic combinations of mutations that improve RNA purity without compromising transcriptional efficiency. We conclude by discussing the remaining challenges for engineering T7 RNAP to meet the stringent purity requirements of next-generation RNA therapeutics.