Genetically Encoded Nucleic Acid Control Architectures

几千年来，人类一直在利用自然微生物的力量。最近，天然系统已经被设计成生产用于制药、能源、化妆品、医疗和其他部门的分子。随着我们设计和控制生物系统的能力的增强，细胞和自主的人工系统已经成为一个目标，这些系统被设计成感知、执行计算和安装程序响应。这种系统在医药和工业方面的潜力都是巨大的。我们的工作将打开利用核酸进行分子电路系统工程的大门。目前，实现这一目标的重大障碍仍然存在。实现生化电路比电学电路更具挑战性，尤其是在活细胞中。很难将生化成分设计成特定的相互作用，从而避免意外的相互作用。此外，对细胞来说，生产成分--特别是蛋白质--的成本很高。这一事实限制了可以在单元中实现的电路的复杂性。最后，生物系统具有内在的随机性，必须小心管理。使用DNA和RNA而不是蛋白质的工程电路可能会减少这些问题。核酸是由一系列规则排列的“碱基”组成的长链，它们之间的相互作用比蛋白质更简单，也更容易理解。沃森-克里克碱基配对的选择性--通过碱基之间的互补相互作用结合核酸链--允许高度特异和基本上任意的相互作用的系统设计。此外，RNA水平的电路不需要将蛋白质生产作为其计算周期的一部分，从而减少了细胞的压力。最后，核酸系统的多功能性和可设计性将使反馈控制系统的模块化和系统化发展成为可能，这些反馈控制系统可以响应和缓解随机波动。要利用RNA实现任意反应，必须通过碱基配对产生多个单链的复合体。这些复合体充当“门”，输入链与之结合，输出链被释放。到目前为止，在混合所有门和电路的所有其他组件之前，每个门都是通过混合其组件链来单独生产的。在一个持续运行、自给自足的系统中，这种两阶段的程序是不切实际的。我们提出了一种直接从单个人工DNA模板产生多链RNA门的机制。通过复制DNA序列产生的单链RNA将折叠成一种结构，在几个位置切割自己的脊椎，从而产生所需的多链门。我们将首先演示基本原理，然后使用这项技术在体外和细胞内实现正和负分子反馈电路。这些反馈主题是控制电路的基本元素，并将展示我们方法的一般性。实验研究将以详细的理论研究为指导，探索以这种方式实现电路的基本原理，在这种方式下，元件不断生产和降解，仍有可能发生意外反应。理论工作还将证明这些电路的可预测性，这些电路是开发合成生物学通用框架的关键组件。最后，我们将展示这些基于RNA的计算电路可以通过将它们耦合到生产荧光蛋白来影响活细胞的行为。这一成果将为这种电路的实际应用打开大门，例如精确控制任何感兴趣基因的表达。从长远来看，它为可预测和系统地开发复杂的人工细胞系统奠定了基础，例如，这种系统可以以一种编程的、但自主的方式对血液中特定疾病相关分子的水平做出反应。

Humans have harnessed the power of natural microbes for millennia. Recently, natural systems have been engineered to produce molecules of use for the pharmaceutical, energy, cosmetic, medical and other sectors. As our ability to design and control biological systems has grown, cells and autonomous artificial systems engineered to sense, perform computations and mount a programmed response, have become a goal. The potential of such systems, in both medicine and industry, is enormous. Our work will open the door to systematic engineering of molecular circuits using nucleic acids.Significant barriers to this goal currently exist. It is much more challenging to implement a biochemical circuit than an electrical one, particularly in living cells. It is difficult to design biochemical components to interact specifically, avoiding unintended interactions. Moreover, producing components - particularly proteins, as is typical - is costly for cells. This fact limits the complexity of circuits that can be implemented in cells. Finally, biological systems have an inherent randomness that must be carefully managed.Engineering circuits using the nucleic acids DNA and RNA, rather than proteins, can potentially reduce these problems. Nucleic acids, long strands with a sequence of regularly-spaced "bases", have simpler and better-understood interactions than proteins. The selectivity of Watson-Crick base pairing - nucleic acid strands bind through complementary interactions between bases - permits the systematic design of highly-specific and essentially arbitrary interactions. Moreover, RNA-level circuits would not require protein production as part of their computational cycle, reducing the strain on the cell. Finally, the versatility and designability of nucleic acid-based systems will enable the modular and systematic development of feedback control systems that respond to and mitigate random fluctuations.To implement arbitrary reactions using RNA, one must produce complexes of multiple single strands bound together through base pairing. These complexes act as "gates" to which input strands bind and from which output strands are released. Hitherto, each gate has been produced separately by mixing its component strands, before mixing all gates and all other components of a circuit. This two-stage procedure is impractical in a continuously-operating, self-sustaining system. We propose a mechanism to produce multi-stranded RNA gates directly from a single artificial DNA template. The single strand of RNA produced by copying the DNA sequence will fold into a structure that cleaves its own backbone in several locations, resulting in the desired multi-stranded gate.We will first demonstrate the basic principle, before implementing positive and negative molecular feedback circuits using this technology, both in vitro and in cells. These feedback motifs are fundamental elements of control circuitry, and will demonstrate the generality of our approach. Experimental studies will be guided by detailed theoretical investigations that explore the fundamental principles of implementing circuits in this way, where components are continuously produced and degraded and unintended reactions remain possible. The theoretical work will also demonstrate the predictability of these circuits, a key component of developing a general framework for synthetic biology.Finally, we will show that these RNA-based computational circuits can influence behaviour in living cells via coupling them to the production of a fluorescent protein. This achievement will open the door to practical applications of this circuitry, such as precisely controlling the expression of any gene of interest. In the long term, it lays the groundwork for the predictable and systematic development of sophisticated artificial cellular systems that could, for example, respond to the level of a particular disease-related molecule in the blood in a programmed, yet autonomous, way.