逻辑门是指一个输入控制一个期望输出的集成系统。在哺乳动物细胞中使用逻辑门是复杂的可逆细胞控制的一个快速发展的领域，具有重要的生物技术和生物医学应用，包括新型传感器、诊断学和治疗学等。特别是，响应小分子的逻辑门可以提供(时空)时间控制，允许对指定的表型进行有针对性的操作。具体来说，对内源性细胞事件没有影响的生物惰性分子是控制逻辑门的理想分子开关，因此能够在细胞中构建真正的正交电路和人工调节通路。本文作者采用生物惰性（非天然氨基酸）来作为逻辑门的输入，这些氨基酸是人工合成的分子，在体外或体内均不产生可观察的表型或毒性。更重要的是，这些非天然氨基酸可以通过基因密码扩展技术，重新利用细胞翻译机制，在哺乳动物细胞中特异性地结合到蛋白质中。
利用正交的氨酰基-tRNA合成酶/tRNA对引入非天然氨基酸具有广泛的应用。然而，每个正交tRNA都需要一个空白(正交)密码子。虽然琥珀终止密码子通常被用作空白密码子，但使用终止密码子作为空白密码子限制了单个细胞中不同的非天然氨基酸引入到蛋白质的数量。为了克服这一局限性，作者评估了11个Pyl tRNA突变体在哺乳动物细胞中解码四联体密码子的能力。有了高效的四元译码正交tRNA突变体，就有可能在哺乳动物细胞中同时引入超过三种不同的非天然氨基酸。
图1 通过遗传密码引入非天然氨基酸的原理
作者构建了11个Pyl tRNAxxxx变体和mCherry-P2A-eGFP(150XXXX)报告基因，其中eGFP的第150个氨基酸残基对应一个四联体密码子，用于测试PylRS/ tRNAxxxx引入的BocK效率的报告基因。不加入BocK会导致翻译终止，eGFP荧光也不会出现。用该载体瞬时转染HEK293细胞，并在加入和不加入1mM BocK的情况下孵育。然后进行流式细胞术分析，并使用图2D显示的公式计算非天然氨基酸的引入效率。其中Pyl tRNACUAG和Pyl tRNAUCCU(Ev2) 引入BocK效果较好。最终作者选用PylRS/Pyl tRNAUCCU(Ev2)引入BocK作为其中一个逻辑门开关。
图2 哺乳动物细胞中用于四元解码的Pyl tRNA突变体
接着，作者又测试了TyrRS*/Tyr tRNACUA正交对。据报道，TyrRS*能够将一系列非天然氨基酸，包括O-甲基- l-酪氨酸(OMeY)和4-叠氮- l-苯丙氨酸(AzF)引入到哺乳动物细胞的蛋白质中。作者发现TyrRS*/Tyr tRNACUA正交对引入AzF的效率优于引入OMeY，所以作者选取TyrRS*/Tyr tRNACUA正交对引入AzF作为逻辑门的另一个开关。并且作者发现，当增加tRNA的拷贝数时，引入非天然氨基酸的效率显著增加。
图3 第二个正交对TyrRS*/tRNACUA
作者引用上述两种正交对引入两种非天然氨基酸，BocK和AzF，并构建了AND和OR的逻辑门。逻辑门的设计采用分离的GFP系统，该系统需要两个互补的多肽sGFP(1-10)和sGFP(11)同时存在形成荧光复合物，当单独产生时，GFP发色团不能组装，也不能检测到荧光。简而言之，作者设计的逻辑门包含两个输入（BocK和AzF）和一个输出（eGFP）。作者用两个载体瞬时转染HEK293细胞，其中一个载体编码TyrRS*/tRNACUA对和mCherry-P2A-TAG-sGFP(1-10)，而另一个载体编码PylRS/tRNAUCCU(Ev2)和mCherry-P2A-AGGA-sGFP(11)。如预期的那样，只有在同时存在AzF和BocK的情况下才可以观察到绿色荧光的细胞，而GFP荧光水平表明该操作的效率是较为理想的（AND逻辑门）。与此类似，作者设计了仅存在AzF或BocK或者两者都存在的情况下均可以观察到绿色荧光的细胞的逻辑门（OR）逻辑门。
图4利用遗传密码扩展构建哺乳动物细胞AND和OR逻辑门
总之，作者评估了HEK293中11个四联体解码的tRNAs引入非天然氨基酸的效率，其中两个tRNAs表现出较高的引入效率。基于此，作者构建了响应两种不同非天然氨基酸的逻辑门，这些发现为哺乳动物细胞逻辑操作提供了一种可替代的方法。
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