工程化改造T7 RNA聚合酶用于高纯度体外转录
Engineering T7 RNA Polymerase for High-Purity In Vitro Transcription
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中文总结 AI 辅助
研究利用噬菌体T7 RNA聚合酶进行体外转录时产生副产物影响RNA生产的问题，通过综合T7 RNAP副产物形成的知识，回顾提高RNA纯度的工程策略，分类T7 RNAP变体，确定权衡原则，突出协同突变组合，为下一代RNA治疗解决纯度挑战。
中文摘要 AI 辅助
使用噬菌体T7 RNA聚合酶（T7 RNAP）的体外转录是研究和治疗应用中RNA生产的金标准平台。尽管T7 RNAP具有高持续合成能力和启动子特异性，但会产生多种RNA副产物，降低RNA产量，使下游纯化复杂化，并引发安全问题。虽然反应优化和下游纯化策略可缓解这些问题，但通常涉及RNA纯度和产量的权衡。因此，酶工程已成为一种强大的上游策略，可从分子源头抑制副产物形成。本文综合了T7 RNAP副产物形成的结构和机制基础的现有知识，并系统回顾了提高RNA纯度的工程策略。T7 RNAP变体根据其潜在作用机制分类，包括增强热稳定性、减少非特异性模板结合、使起始到延伸的过渡更顺畅、减少过早终止以及模板偏向的聚合酶设计。该分析确定了特异性和持续合成能力之间权衡的一般原则，并突出了在不影响转录效率的情况下提高RNA纯度的突变协同组合。最后讨论了工程化改造T7 RNAP以满足下一代RNA治疗严格纯度要求的剩余挑战。
英文摘要
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.
