Development of mammalian cell logic gates controlled by unnatural amino acids.
Development of mammalian cell logic gates controlled by unnatural amino acids.
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DOI：
10.1016/j.crmeth.2021.100073
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发表时间：
2021-10-25
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哺乳动物细胞逻辑门具有广泛的应用潜力。然而，目前可用的大多数是由具有固有生物活性的药物（类）分子控制的。为了构建真正的正交电路和人工调控途径，生物惰性分子是理想的分子开关。在这里，我们应用了遗传密码扩展和工程逻辑门控制的两个生物惰性的非天然氨基酸。遗传密码扩增依赖于正交氨酰-tRNA合成酶/tRNA对，用于共翻译和位点特异性非天然氨基酸掺入，通常响应于琥珀（UAG）密码子。通过从文献中筛选11个四联体解码吡咯赖氨酰tRNA变体，我们发现这里测试的所有解码CUAG或AGGA的变体在哺乳动物细胞中都是功能性的。利用四重解码正交对和琥珀解码对，我们构建了可以成功地由两种不同的非天然氨基酸控制的逻辑门，扩展了遗传密码扩展和哺乳动物细胞逻辑电路的范围。我们在HEK 293中评估了11个四联体密码子解码tRNA的性能。两个tRNA在四联体密码子解码中显示出高效率。我们构建了响应于两个不同的非天然氨基酸的逻辑门。这些发现为哺乳动物细胞的逻辑操作提供了一种替代方法。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用作空白密码子，但使用终止密码子作为空白密码子不可避免地限制了在单个细胞中掺入蛋白质中的可能的不同非天然氨基酸的数量。为了克服这一限制，我们评估了11个Pyl tRNA变体在哺乳动物细胞中解码四联体密码子的能力。随着有效的四联体解码正交tRNA变体的可用，可以在哺乳动物细胞中同时掺入三种以上不同的非天然氨基酸。逻辑门能够通过小分子调节蛋白质功能，但这些小分子通常是具有内在生物活性的药物（类）分子。米尔斯等人通过遗传密码扩展提出了一种替代方法，表明非天然氨基酸可以作为有效的哺乳动物细胞逻辑操作的生物惰性开关。
Mammalian cell logic gates hold great potential for wide-ranging applications. However, most of those currently available are controlled by drug(-like) molecules with inherent biological activities. To construct truly orthogonal circuits and artificial regulatory pathways, biologically inert molecules are ideal molecular switches. Here, we applied genetic code expansion and engineered logic gates controlled by two biologically inert unnatural amino acids. Genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase/tRNA pairs for co-translational and site-specific unnatural amino acid incorporation conventionally in response to an amber (UAG) codon. By screening 11 quadruplet-decoding pyrrolysyl tRNA variants from the literature, we found that all variants decoding CUAG or AGGA tested here are functional in mammalian cells. Using a quadruplet-decoding orthogonal pair together with an amber-decoding pair, we constructed logic gates that can be successfully controlled by two different unnatural amino acids, expanding the scope of genetic code expansion and mammalian cell logic circuits. We evaluate the performance of 11 quadruplet-decoding tRNAs in HEK293 Two tRNAs show high efficiency in decoding quadruplet codons We construct logic gates responding to two different unnatural amino acids These findings present an alternative approach to mammalian cell logic operations Genetic code expansion employing an orthogonal aminoacyl-tRNA synthetase/tRNA pair for site-specific unnatural amino acid incorporation has a wide range of applications. However, a blank (orthogonal) codon is needed for each orthogonal tRNA. Although the amber stop codon is commonly used as the blank codon, the use of stop codons as the blank codons inevitably limits the number of possible different unnatural amino acids to be incorporated into proteins in a single cell. To overcome this limitation, we evaluated the capability of 11 Pyl tRNA variants for decoding quadruplet codons in mammalian cells. With efficient quadruplet-decoding orthogonal tRNA variants available, it is possible for simultaneous incorporation of more than three different unnatural amino acids in mammalian cells. Logic gates enable regulation of protein function by small molecules, but these are often drug(-like) molecules with intrinsic biological activities. Mills et al. present an alternative approach through genetic code expansion, demonstrating that unnatural amino acids can act as biologically inert switches for effective mammalian cell logic operations.