The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg²⁺ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated.

中文翻译：

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体外转录RNA免疫原性的起源


基于 RNA 的疗法的出现需要稳健且经济的方法来生产 RNA，并且几乎不产生异常活性的副产物。虽然使用噬菌体 T7 RNA 聚合酶进行体外转录是一种流行的方法，但众所周知，其转录物显示出通常不受欢迎且无法控制的免疫刺激活性。我们在此表明​​，T7 转录物的免疫刺激活性是由其从无启动子的 DNA 末端启动转录的异常活性促成的。这种活性导致产生与预期有义 RNA 产物完全互补的反义 RNA，从而产生能够强烈刺激胞质模式识别受体 MDA5 的长双链 RNA (dsRNA)。 T7 RNA 聚合酶的这种独立于启动子的转录活性在多种 DNA 序列和长度中都被观察到，但可以通过用修饰的核苷酸改变转录反应或通过降低 Mg ²⁺浓度来抑制。因此，目前的工作不仅提供了一种以前未被认识到的 T7 转录物刺激先天免疫系统的机制，而且还表明免疫刺激活性可以很容易地调节。