Genetically Encoded Nucleic Acid Control Architectures

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.

Impact on society: The EPSRC is a strong supporter of synthetic biology, and plans to increase funding further due to its "potential to be transformational in a large number of key application areas." Similarly, the Department for Business, Information and Skills named synthetic biology as one of the "eight great technologies that support UK science strengths and capabilities." Our project will produce a new paradigm for implementing modular, predictable, economical and robust biochemical circuits in cells, and a deeper understanding of the fundamental principles that determine the effectiveness of such biochemical circuits. These outcomes are central to fulfilling the transformative potential of synthetic biology, and our project will therefore contribute strongly to its future growth in the UK.

The EPSRC has also identified "Grand Challenges" that will yield significant societal impact. The "Systems Chemistry" Grand Challenge highlights the importance of exploiting molecular reactions to create new functions and systems, including those that process information. Our project, using nucleic acids to develop genetically-encoded circuits that respond to complex internal and external signals, fits naturally into this framework, both within the in vitro and in vivo contexts. Given the scope for general interest, the project is also a natural candidate for the development of research-based public engagement and outreach activities.

Impact on economy and industry: Our main target for economic and industrial impact is the rapidly developing industry surrounding synthetic biology. The EPSRC has identified genuinely modular design as a key requirement for "advancing the field [of synthetic biology] to commercialisation". Further, the ability to refine systems through fine-tuning is central to producing commercially viable outputs, as is the need to reduce the burden on the cell hosting the synthetic circuitry. Our work meets these challenges by paving the way for exquisitely modular, adjustable and low-cost nucleic acid circuits in cells. Our approach is also a natural basis for the development of stochastic biochemical controllers, as we will demonstrate. Artificial control of biological systems is a key step towards enhancing robustness in industrial processes, and the eventual development of autonomous, decision-making cellular systems.

A secondary target is in vitro nucleic acid computation, which is actively researched by companies such as Microsoft. Nucleic acid systems are unlikely to displace silicon, but they are highly parallel and can interface directly with chemical and biological signals. Our work will provide a new paradigm within which to perform nucleic acid computation, in which systems can function robustly and autonomously due to the continuous production and degradation of components.

Impact on knowledge and training: Our work will facilitate the exchange of ideas between biology, engineering and physical sciences. At these interfaces, theoretical principles suggest new experimental approaches, and experimental realities provide inspiration to deepen understanding of the principles. Those working at this interface develop a broad set of experimental and mathematical skills, essential to the development of a successful UK bioeconomy.

At the highest level, our project will facilitate collaboration across this interface by making synthetic biology more accessible to engineers and physical scientists. More directly, the work will be undertaken collaboratively by two RAs with backgrounds in biological and physical sciences, overseen by PIs with expertise in control engineering, synthetic biology and physics. Through collaborations, related undergraduate and graduate projects, and the incorporation of outputs into teaching, the work will have impact across the UK's leading bioengineering department at Imperial.