Engineering protein activity into off-the-shelf DNA devices.
Engineering protein activity into off-the-shelf DNA devices.
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DOI：
10.1016/j.crmeth.2022.100202
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发表时间：
2022-04-25
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基于DNA的装置由于其可预测的折叠而易于设计，但它们缺乏复杂的生物活性，如催化。相反，基于蛋白质的设备提供了无数的功能，但由于其复杂的折叠而更难以设计。这项研究结合了DNA和蛋白质工程，以产生一种由选择的DNA序列激活的酶。使用交替框架折叠机制从纳米荧光素酶工程化的并且在本文中称为nLuc-AFF的单个蛋白质开关与不同的DNA技术配对，以创建用于特定核酸序列的生物传感器、用于血清素和ATP的传感器以及双输入逻辑门。nLuc-AFF是一种基因编码的比率式蓝/绿发光生物传感器，其输出可通过手机摄像头进行量化。nLuc-AFF在100%血清中保留比率读数，使其适用于在低资源环境中分析粗样品。这种方法可以应用于其他蛋白质和酶，将它们转化为DNA激活的开关。nLuc-AFF是一种发光蛋白开关，其颜色由DNA输入控制nLuc-AFF在血清中起作用，其输出由手机摄像头定量传感器输出由DNA序列、逻辑门和适体控制技术可以潜在地将其他蛋白质转化为DNA/RNA激活的开关自然界给了我们一个蛋白质的例子，Cas酶，通过结合特定的DNA或RNA序列来激活。Cas酶具有切割核酸的独特功能，但它们已经产生了改变生物学的基因编辑技术。将这种控制模式引入其他蛋白质和酶中尚不可行。在这里，我们融合的DNA结合结构域（GCN 4）的酶（nanoluciferase），使特定的DNA序列结合到前者触发后者的构象变化，导致比率绿色到蓝色的发光变化。这种蛋白质开关插入现有的DNA工具，以生成用于选择的DNA和RNA序列以及小分子和蛋白质的生物传感器。这种机制是通用的，基于蛋白质折叠和核酸碱基配对的既定原则，并有可能通过核酸和其他生物分子来调节蛋白质组的生物活性。Sekhon和洛开发了一种发光蛋白质开关，该开关响应于各种DNA输入而经历构象变化，将其颜色从绿色转换为蓝色。这种蛋白质与DNA工程技术配对，以检测特定的DNA序列和小分子，并处理双输入逻辑门。
DNA-based devices are straightforward to design by virtue of their predictable folding, but they lack complex biological activity such as catalysis. Conversely, protein-based devices offer a myriad of functions but are much more difficult to design due to their complex folding. This study combines DNA and protein engineering to generate an enzyme that is activated by a DNA sequence of choice. A single protein switch, engineered from nanoluciferase using the alternate-frame-folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for specific nucleic acid sequences, sensors for serotonin and ATP, and a two-input logic gate. nLuc-AFF is a genetically encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by a phone camera. nLuc-AFF retains ratiometric readout in 100% serum, making it suitable for analyzing crude samples in low-resource settings. This approach can be applied to other proteins and enzymes to convert them into DNA-activated switches. nLuc-AFF is a luminescent protein switch whose color is controlled by DNA inputs nLuc-AFF is functional in serum, and its output is quantified by phone camera Sensor output is controlled by DNA sequences, logic gates, and aptamers Technique can potentially convert other proteins to DNA/RNA-activated switches Nature has given us an example of proteins, the Cas enzymes, that are activated by binding specific DNA or RNA sequences. Cas enzymes possess the lone function of cleaving nucleic acids, yet they have given rise to a gene-editing technology that has transformed biology. It has not yet been feasible to introduce this mode of control into other proteins and enzymes. Here, we fuse a DNA-binding domain (GCN4) to an enzyme (nanoluciferase) such that binding a specific DNA sequence to the former triggers a conformational change in the latter, resulting in a ratiometric green-to-blue luminescence change. This protein switch plugs into existing DNA tools to generate a biosensor for DNA and RNA sequences of choice, as well as for small molecules and proteins. This mechanism is general—based on established principles of protein folding and nucleic acid base pairing—and has the potential to access the biological activity of the proteome to regulation by nucleic acids and other biomolecules. Sekhon and Loh develop a luminescent protein switch that undergoes a conformational change in response to various DNA inputs, switching its color from green to blue. This protein is paired with DNA engineering techniques to detect specific DNA sequences and small molecules and process two-input logic gates.