A team of biologists at Harvard’s Wyss Institute for Biologically Inspired Engineering fit cells with artificial molecular machinery that could sense stimuli like toxins in the environment, metabolite levels and inflammatory signals. The circuits it creates work similar to electronic currents; they process information and make logic-guided decisions. The difference between the electronic circuits and the biological circuits is the biological ones have to be made from molecular components that cells make and the need to be able to operate in crowded and changing environments within each cell.
The synthetic biological circuits have so far been able to sense a handful of signals which gives them an incomplete idea of conditions in host cells. The circuits also also built using several moving parts like different molecules of DNAs, RNAs and proteins. The molecules have to bind and work together to sense and process signals.
Biological circuits are generally hard to make because it is difficult to find molecules that work well with one another. The process can sometimes be time-consuming and unpredictable. The Wyss Institute researchers have figured out a solution that imbues an RNA molecule with the capacity to sense multiple signals and make logical decisions that help control protein production with precision.
The approach resulted in a genetically encodable RNA nano-device that performs 12-input logic operation to regulate expressions of fluorescent reporter protein in E. coli bacteria only when it comes across a complex, user-prescribed profile of intra-cellular stimuli. Programmable nano-devices like this could allow researchers to make more sophisticated synthetic biological circuits to analyze complex cellular environments.
“We demonstrate that an RNA molecule can be engineered into a programmable and logically-acting ‘ribocomputing device,’” Peng Yin, Wyss Institute Core Faculty member and leader of the study, said in a press release. “This breakthrough at the interface of nanotechnology and synthetic biology will enable us to design more reliable synthetic biological circuits that are much more conscious of the influences in their environment relevant to specific goals.”
The team created what they called toehold switches in 2014 that were programmable hairpin-like nano-structures made of RNA. The switches were able to control a specific protein’s production when complementary trigger RNA binds to the toehold switch to break open the hairpin structure. The cell’s ribosomes could then access the RNA and produce desired proteins. This process was the inspiration behind their new ribocomputing devices.
“We wanted to take full advantage of the programmability of toehold switches and find a smart way to use them to expand the decision-making capabilities of living cells. Now with ribocomputing devices, we can couple protein production to specific combinations of many different input RNAs and only activate production when conditions allow it,” Alexander Green, co-first and corresponding author, said. “Once we had worked out how to use Toehold Switches and RNA molecules to encode the basic logic operations – and, or, and not – we were able to condense this functionality within a carefully designed molecule that we call a gate RNA. Use of a gate RNA makes the ribocomputing devices much more genetically compact and helps with scaling up the circuits so that the cells can make more complex decisions.”
The researchers suggest that the ribocomputing devices could be used in cell-free applications in addition to different living organisms.
“These logic-based RNAs could be freeze-dried on paper and thus boost the possibilities of paper-based biological circuits, including diagnostics that can sense and integrate several disease-relevant signals in a clinical sample,” James Collins, the study’s co-author, said.
The research was funded by the Wyss Institute’s Molecular Robotics Initiative, Defense Advanced Research Projects Agency Living Foundries, National Institutes of Health, the Office of Naval Research, the National Science Foundation and the Defense Threat Reduction Agency. It was published in the Nature journal.