‘upgraded’ by making miRNA activity the outcome of proportional or inverse sensing of external inputs. For example, if genetically encoded synthetic miRNAs are activated or repressed by transcription factors, any logic formula can in principle be computed with these transcription factor inputs.
反式作用RNA开关包括高等真核生物中细菌和微分子RNA中的小分子RNA（sRNA）。miRNAs被广泛研究作为在哺乳动物细胞中复杂的逻辑基础，因为多个miRNAs控制相同的基因，实现或非逻辑。逻辑可以通过让miRNA的活性比例输出或逆传感外部输入改善。例如，如果基因编码合成的miRNA通过转录因子激活或抑制，原则上任何逻辑公式都可以用这些转录因子输入进行计算。
Recently, a hybrid system was constructed that interrogates six endogenous miRNA markers to identify HeLa cancer cells. The circuit computes an AND logic with these markers, detecting a HeLa-specific expression profile and triggering an apoptotic gene in response (FIG. 5b). In contrast to miRNAs, sRNAs have yet to be extensively used in circuit design, despite the fact that natural circuits with complex sRNA-based logic have been discovered and simple synthetic switches have been built.
最近，构造了一种混合系统，通过审查六个内源性miRNA标记，来识别HeLa癌细胞。该电路通过这些标记实现了与逻辑计算，检测HeLa细胞特异的外形表达，响应触发细胞凋亡基因（图5b）。与miRNAs不同的是，sRNA已经被广泛应用于电路设计，尽管事实上，基于复杂的sRNA逻辑的自然电路已被发现，简单的合成开关也已建成。
图 5-b
Protein-based biochemical circuits 基于蛋白质的生物分子电路 In biochemical systems, protein enzymes have taken the central role because they can be used to implement a ‘metabolic logic’, in which the inputs and the outputs are enzyme substrates and products. Examples include a network of coupled enzymes with four substrate inputs that computed a four-input logic function and a system that implemented a set of universal NOR and NAND gates. Extensive theoretical analysis has suggested ways of coping with noise and uncertainty in enzymatic circuits. These results point to new ways of controlling metabolism by computational integration of key intermediates. In a
different effort, self-assembling peptides were extensively investigated as building blocks for logic networks. Translating these approaches to cells may open new avenues in protein-based biological computing.
生化系统中，蛋白质酶已经担任了核心的角色，因为它们可以被用来实现一个“代谢逻辑”，他的输入和输出都是酶的底物和产物。例子包括具有四个基板输入的耦合酶素的网络，用于计算四输入的逻辑函数和实现一套通用的或非、与非门。广泛的理论分析提出了用酶电路处理噪声和不确定度。这些结果指出了用计算合成关键中间体控制新陈代谢的新途径。在不同的工作中，自组装肽被广泛研究作为逻辑网络的构建模块。将这些途径转译到细胞可能开辟基于蛋白质的生物计算的新渠道。
Protein-based biological switches and circuits 基于蛋白质的生物转换开关和电路
Most engineered protein circuitry in cells uses transcription factors. Because they are very well understood, transcription factors have long been the subjects of rational design and are currently the workhorses of circuit engineering. In addition to a large repertoire of native transcription factors, de novo switches can be engineered from recombinant transcription factors that combine protein domains from different organisms and their target promoters. 大多细胞蛋白电路设计使用转录因子。因为他们很好理解，转录因子一直是合理设计的核心，是目前电路工程的骨干。除了大部分的本地转录因子，更使开关可以从重组转录因子中设计，从不同的生物体和他们的目标启动子中结合蛋白结构域。
Transcriptional activation and repression are usually interpreted as ‘buffer’ gates (equivalency) and NOT gates, respectively. In addition, circuits
combining a number of transcriptional elements have been shown to implement analogue bandpass behaviour in response to a diffusing chemical, with the output produced at intermediate, but not at low or high, input levels. In multiple-input systems, two-input promoter logic can comprise OR, NOR and ‘AND NOT’ operations but rarely AND. This is because AND would require a hard-to-engineer interaction between two transcription factors.
转录激活和抑制通常被分别看作是“缓冲”门和非门。此外，结合一部分转录元件的电路已被证明，通过对扩散化学的响应模拟带通滤波，在不高不低的输入电平下，输出中间产物。在多输入系统，两个输入启动子的逻辑可以包括或，或非，与非运算，但很少出现与运算。这是因为与运算需要两个转录因子之间的相互作用，这对工程师来说都是很难的工作。
However, a further increase in complexity to three or more inputs in a rational fashion has proved to be refractory, apart from in a few examples that are based on rational design and promoter library screening. Therefore, most experimental transcriptional circuits use one- or two-input universal gates in parallel and in cascades to increase the total number of inputs (FIG. 6a). 然而，除了在少数的基于合理的设计和启动子文库筛选的例子中，合理的将复杂性扩展到三个或更多输入已被证明是难治性的。因此，大多数实验的转录电路通过一个或两个输入的通用门的并联和级联来增加总输入数（图6a）。
图 6-a
Transcription factors can serve as inputs to the logic circuits or as sensors of other molecules or agents that modulate their activity. So far, in most reported biological computing circuits, they have been used as transducers of small-molecule concentrations. In addition, light sensing was used in a bacterial circuit to implement an edge detection task and in mammalian cells to activate a therapeutic gene product. These examples point to a potential use