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.
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