The 5′ untranslated region (5′UTR) plays a key role in post-transcriptional regulation, but interaction between nucleotides and directed evolution of 5′UTRs as synthetic regulatory elements remain unclear. By constructing a library of synthesized random 5′UTRs of 24 nucleotides in Saccharomyces cerevisiae, we observed strong epistatic interactions among bases from different positions in the 5′UTR. Taking into account these base interactions, we constructed a mathematical model to predict protein abundance with a precision of R² = 0.60. On the basis of this model, we developed an approach to engineer 5′UTRs according to nucleotide sequence activity relationships (NuSAR), in which 5′UTRs were engineered stepwise through repeated cycles of backbone design, directed screening, and model reconstruction. After three rounds of NuSAR, the predictive accuracy of our model was improved to R² = 0.71, and a strong 5′UTR was obtained with 5-fold higher protein abundance than the starting 5′UTR. Our findings provide new insights into the mechanism of 5′UTR regulation and contribute to a new translational elements engineering approach in synthetic biology.

中文翻译：

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利用核苷酸序列活性关系设计酵母中5'UTR介导的蛋白质丰度调节

5'非翻译区（5'UTR）在转录后调控中起关键作用，但由于合成调控元件，核苷酸之间的相互作用和5'UTR的定向进化尚不清楚。通过构建酿酒酵母中24个核苷酸的随机5'UTR合成文库，我们观察到了5'UTR中不同位置碱基之间的强上位相互作用。考虑到这些基本相互作用，我们构建了一个数学模型来预测蛋白质丰度，精确度为R ²= 0.60。在此模型的基础上，我们开发了一种根据核苷酸序列活性关系（NuSAR）设计5'UTR的方法，其中5'UTR通过骨架设计，定向筛选和模型重建的重复循环逐步进行工程设计。经过三轮NuSAR，我们的模型的预测准确性提高到R ² = 0.71，并且获得了强大的5'UTR，蛋白质丰度比起始5'UTR高5倍。我们的发现为5'UTR调控机制提供了新见解，并为合成生物学中的新翻译元件工程方法做出了贡献。