Working in the labs of Peng Yin and James Collins of the Wyss Institute for Biologically Inspired Engineering at Harvard University, Green already had the potential technologies in hand: customizable RNA-based sensors that he and his colleagues had put on pocket-sized strips of paper.
“It opens up new possibilities in the field by basically making it easier to move synthetic biology out of the lab and into different settings, including the clinic and global health. So we’re very excited about what all this could mean and bring,” said senior author Collins, one of the founders of the field of synthetic biology.
This past summer, synthetic biologist Julius Lucks of Cornell University, who was not involved with the new research, was sitting in the audience at the inaugural Synthetic Biology, Engineering, Evolution and Design (SEED) meeting in Los Angeles when Collins presented the work. “Everyone wants to engineer cells because cells do amazing things, and this paper is saying, ‘I’m going to take all the amazing things in a cell and put them on a piece of paper.’ It just simplifies everything,” said Lucks. “I know I wasn’t the only one in the audience thinking, ‘Damn, why didn’t I think of that?’”
The Key Steps
One key piece of technology in the diagnostic is an RNA-based biological part called a “toehold switch” that can detect and visualize any sequence of RNA. The first generation of the toehold switch was born about two years ago, when Green created a piece of RNA designed to activate the expression of any specific gene in E. coli—for example, the gene for a fluorescent protein or an enzyme-based colorimetric reaction—when bound to an RNA sequence of choice.
Green and his colleagues compared the viral sequences of the Sudan and Zaire Ebola virus strains and identified short, conserved stretches of RNA that could be used to tell the two apart. They ordered the bits of sequence and quickly designed 24 new toehold switches that could distinguish the strains.
The tools will be useful for constructing many other synthetic gene networks because the reactions work within a large range of RNA concentrations and show good orthogonality, meaning that multiple switches can switch on independent activities within a cell. “We show in the paper that we can have 12 different toehold switches active in a cell and independently regulating different proteins,” Green said.
Synthetic biologists typically harness whole cells to do their bidding, to churn out particular proteins, RNA, or initiate signaling cascades. But the problem with cells is two-fold, said Keith Pardee, postdoctoral fellow in the Collins lab and first author of the second paper (2): First, the cell wall forms a protective layer that makes it more difficult for plasmids to enter. The second obstacle is that the introduction of a genetic construct requires the use of antibiotic resistance as a selection marker, which becomes unwieldy as you build more complex systems.
In contrast, cell-free extracts, which contain all of the crucial transcription and translation machinery of cells without the cell membranes, don’t come with such concerns about cells walls and antibiotic resistance. “It’s more like programmable chemistry than it is like cell-based biology,” Pardee said. In addition, going cell-free is an attractive choice, because it avoids the ethical considerations of engineering a live organism.
But according to Pardee, such reactions are generally awkward to handle outside the lab. Inspired by the chemistry-based diagnostics that George Whitesides, also of the Wyss Institute, had put on paper, Pardee proposed doing the same with toehold switches.
When that worked, Pardee set about to eliminate the need for refrigeration. His idea was to freeze dry the paper. “At the beginning, it wasn’t intuitive that it was going to work,” he said. “There are at least 35 proteins involved, and to be able to get them all to reconstitute and work in concert—I didn’t think it was going to work, but it did.”
Paper-based tests are cheap, typically costing under $1 per test to manufacture. But running the test and quantifying the result are two different processes. The new paper-based test results are visible to the naked eye and give a yes-or-no answer; to quantify the colorimetric reactions, the researchers built an optical reader for less than $100.
Several other hurdles must be crossed before these slips of paper become diagnostics, and the team is working on them now. One goal is to couple the platform with the appropriate sample prep technology so that blood, urine, or other bodily fluids are processed appropriately for application to the paper.
The system’s sensitivity also needs further improvement. “Right now we’re in the low picomolar range, and we need to improve that even further for it to be used for many applications. We’re actually looking to see whether we can further harness synthetic biology to do that,” Collins said. In addition, the team is exploring ways to concentrate fluids.
Another challenge is that RNA folding makes some sequences less accessible to toehold switches than others, so Green and others are working on ways to predict RNA structure. “It’s currently a challenge, but we don’t think it will be for long,” Lucks said. His group has recently developed and improved a tool they call SHAPE-Seq for helping overcome this problem (3,4).
And other new tools, such as the Ebola
Genome Browser can be used for designing new toehold switches because
they give information about precisely where the virus is changing; the team
can then pinpoint conserved regions of the genome or transcriptome to target
with the sensors.
1. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014 Nov 6;159(4):925-39. doi: 10.1016/j.cell.2014.10.002. Epub 2014 Oct 23.
2. Pardee K, Green AA, Ferrante T, Cameron DE, DaleyKeyser A, Yin P, Collins JJ. Paper-based synthetic gene networks. Cell. 2014 Nov 6;159(4):940-54. doi: 10.1016/j.cell.2014.10.004. Epub 2014 Oct 23.
3. Lucks JB, Mortimer SA, Trapnell C, Luo S, Aviran S, Schroth GP, Pachter L, Doudna JA, Arkin AP. Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci U S A. 2011 Jul 5;108(27):11063-8. doi: 10.1073/pnas.1106501108. Epub 2011 Jun 3.
4. Loughrey D, Watters KE, Settle AH, Lucks JB. SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing. Nucleic Acids Res. 2014 Dec 1;42(21):0. doi: 10.1093/nar/gku909. Epub 2014 Oct 10.