Sometimes the biggest challenge for a researcher is to ignore nature's tested and tried ways. Peng Yin and his collaborators at Harvard University recently shared this experience when looking to expand the number of RNA-based regulatory components for synthetic networks.

During the past decade, nature's wide variety of RNA-based transcriptional and post-transcriptional regulators has inspired the design of riboregulators that control transcription and translation in response to an input RNA signal. James Collins, also at Harvard and one of Yin's collaborators, pioneered the design of riboregulators in 2004. By inserting a complementary sequence upstream of the ribosome-binding site (RBS) that formed a stem-loop after transcription, he created a structure that could interfere with ribosome binding. Only the binding of a small noncoding RNA, expressed in trans, to this stem-loop opened the structure and allowed translation. Providing or withholding the RNA trigger could thus regulate the gene.

Although this RNA-based system had advantages over protein-based regulation in that it was easier to design and exerted less of a burden on cells, its dynamic range—the output over input signal—of up to 50-fold was much lower than that of protein regulators, which can achieve around 500-fold. Another limitation was the lower specificity because of sequence constraints between cis sequences that have to form a secondary structure and their complementary trigger RNAs. About a fifth of all known riboswitches showed cross-talk.

Binding of the trigger RNA to the toehold opens the transcript for translation. Credit: Figure reproduced from Green et al., Elsevier.

Thus, Yin, Collins and their joint postdoctoral fellow Alexander Green decided to design new riboregulators from scratch, basing them on what is known about RNA-RNA interactions rather than on existing examples (Green et al., 2014)1. Their toehold design contains a switch RNA, the gene to be regulated and an upstream hairpin that includes the RBS; but instead of requiring the trigger RNA to bind to this stem-loop, the RNA binds to a linear toehold at the 5′ end of the hairpin, thus allowing far greater design flexibility. Upon binding of the trigger RNA to the toehold, the structure opens up and translation proceeds.

Their first generation of toehold switches included 168 varieties, of which 20 showed a dynamic range of greater than 100 when tested with a GFP reporter in Escherichia coli. “But,” says Yin, “some did not work ... so we empirically tested and distilled critical design parameters.”

The second-generation riboregulators included four design changes: sequence alterations in the stem and loop as well as an increase in length of the toehold sequence and its shift further away from the RBS. The resulting toggle switches showed a 400-fold dynamic range, and only 1 of 13 switches did not work.

Although the group spent almost 2 years on the design, demonstrating different applications took only 4 months. Green used a toehold switch to sense or to regulate endogenous genes. He developed a multiplexed regulatory system in which the expression of four fluorescent proteins is regulated in parallel and designed a four-input AND gate.

The toehold switches also work in vitro. In a recently developed paper-based platform for diagnostics by the Collins group, a circuit containing a toehold switch is dried onto paper and upon rehydration can test for the presence of a trigger RNA (Pardee et al., 2014)2.

Yin's group is now working on toehold switches for mammalian cells.