Biomolecular computing systems principles, progress and pote(5)

subunits that formed functional enzymes only following certain input combinations, implementing complex logic cascades。

因此，天然的建筑模块作为全新开关的基础，确保了他们在细胞内的运作。下面我们描述了基于DNA的开关及电路设计，其次是RNA，然后是蛋白质的建筑模块。由多级子单位组成，形成的功能酶只能遵循一定的输入组合，从而实现复杂的逻辑通路。

A different approach in oligonucleotide-based logic is based on the observation that abstract reaction networks can be instantiated with DNA species interacting through strand migration or strand displacement without creating or breaking chemical bonds. From initial experimental computations with a four-gate circuit integrating six microRNA-like inputs in the test tube, these circuits have grown to include up to twelve gates, eight inputs and more than 100 building blocks.

通过报告分析，发现了一种基于寡核苷酸的不同的逻辑实现，抽象的反应网络可以不新建也不打破化学键，通过链迁移或 链置换和DNA分子相互作用。通过初始的四门电路的实验，在试管里集成六个类似微小核糖核酸的输入，这些电路已发展到多达十二个门，八个输入和100多个构建模块。

These most recent gates are based on artificial neurons with thresholds (FIG. 4), implementing multiple-input–multiple-output AND and OR logic. Molecular neurons were also used to build a circuit resembling a small Hopfield neural network with associative memory features, underlying their use in both digital and analogue computing networks.

最新的门基于带有阈值的人工神经元（图4），实现多输入多输出的与逻辑和或逻辑。分子神经元被用来建立一个具有联想记忆功能的类似小型Hopfield神经网络的电路，其基础应用是数字计算网络和模拟计算网络。

图 4

Gene-based biological switches 基于基因的生物转换开关

Modification of the gene sequence (in the promoter and/or the coding region) not only affects a biological process, but it does so in a heritable fashion. Engineering modifications in the gene sequence can be most readily achieved with site-specific recombinases that are commonly used for genomic manipulation, such as Cre. The modification may include excision, insertion or inversion of a gene fragment, and it can be reversible or irreversible, depending on the recombinase and the arrangement of the recognition sites. 基因序列的修改（在启动子和编码区）不仅影响生化过程，而且它是以一种可遗

传的方式完成这项工作。基因序列的工程改造通过位点特异性是最容易实现的，通常用于基因组的处理，如肌酐。改造工作包括一个基因片段的切除，嵌入和倒置，这些工作可以是可逆，也可以是不可逆的，取决于重组酶和识别位点的排列。 Recombination becomes a truly digital biomolecular process when the cell contains exactly one copy of the genetic substrate and the process is irreversible. Interestingly, bona fide logic circuits with recombinases have yet to be shown. Instead, a number of experimental systems implemented the state machine computations, as we will see below. Another potentially interesting tool for heritable DNA modification is transposable elements: ‘selfish’ genetic elements that encode their own integration machinery.

当细胞恰好包含一个基因基板的拷贝时，重组成为一个真正的数字生物分子进程，其过程是不可逆的。有趣的是，真正由重组酶构成的逻辑电路还未出现。相反，我们下面将看到一些实验系统实现的状态机计算。另一个可能出现的可遗传性DNA修饰的工具是转位因子：只编码自身集成机构的自私的遗传因子。 Epigenetic switching is heritable modification of a gene’s state that occurs without changes in the nucleotide sequence — for example, by DNA methylation or histone modification. The change in state is usually rather binary, if slow. Moreover, the triggers causing the change and the biomolecular mechanisms are still poorly understood.

后生的开关是一个不改变核苷酸序列的基因状态修正的遗传性修饰。例如，通过DNA甲基化和组蛋白的修饰。状态的变化，如果慢的话通常相当于二进制。然而，对于触发器造成的变化及其分子机制仍知之甚少。

Thus, epigenetics have rarely been used in engineered circuits. However, a recent report showed a designed transcription factor that binds specific modi-fied histones and upregulates gene expression at adjacent loci using a VP64

transactivator domain (modified from herpes simplex virus). Large engineered circuits that incorporate rationally designed epigenetic switches might be useful for tissue engineering and cell-based therapy applications.

因此，实验胚胎学很少被运用在电路设计中。然而，最近的一份报告显示了一个结合特定的组蛋白修饰的转录因子的设计，使用反式激活因子域VP64在临近位点上调基因表达（由单纯疱疹病毒进行改动）。包含合理的后生开关的设计的大型工程电路，可能对组织工程和细胞治疗的应用有所帮助。

RNA-based biological switches and circuits 基于RNA的生物转换开关和电路

RNA-centred switches (FIG. 5a) have gained attention in recent years owing to their small size, modularity and susceptibility to diverse inputs such as metabolites, RNA and proteins. A typical bifunctional cis-acting riboswitch controls its host gene by transcriptional termination, ribosome-binding site blockage or self-cleavage; ligand binding modulates the gene control function. Engineered riboswitches have been integrated in tandem to implement various two-input gates.

RNA集中开关（图5a）由于他们的小尺寸，模块性和对不同输入物如代谢物、RNA和蛋白质的敏感性，已经在近几年得到了关注，。一个典型的双官能团的顺式作用的核糖开关，通过转录终止、核糖体结合位点堵塞和自我裂解控制其宿主基因；配体结合调节基因控制。核糖开关的设计被一前一后的集成，实现各种双输入门。

图 5-a

Recent work has shown a two-input NOR gate and a three-level cascade that is controlled by the interaction of an antisense-like RNA input with a riboswitch. Another study described a novel class of RNA aptamer switches that, when placed in intronic location, regulate alternative splicing following binding of cognate endogenous protein inputs。

近期的工作表明，双输入或门非和三级级联是通过带有核糖开关的反义RNA开关输入的互动所控制。另一项研究描述了一种新的RNA适配子开关，当放置在内含子的位置，通过绑定同源的内源蛋白输入选择性剪接调节。

Trans-acting RNA switches include small RNAs (sRNAs) in bacteria and microRNAs (miRNAs) in higher eukaryotes. miRNAs have been extensively studied as the basis for complex logic in mammalian cells, because multiple miRNAs that control the same gene implement NOR logic. The logic can be

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