Freeze-drying has claimed to be the technology the future before, but its fruits – from strawberries to ice cream – have never quite lived up to the hype. However, synthetic gene circuits recently joined the picnic with a powerful new use for the process; they can be freeze dried onto filter paper along with cell-free transcription-translation mixes.. When the mixes are re-hydrated with a drop of water, the gene circuits turn on, and if the water contains a detectable signal, the circuits can sense it and produce a color-based output.
Now, a team from Jim Collins’ lab in Boston has harnessed this technique to make sensitive, portable, low-cost Zika virus sensors. Like many diseases, Zika does not consider regional resources when it decides where to rampage. This makes many detection methods too expensive, and Zika is particularly problematic because similar viruses have antibody cross-reactivity, so genetic analysis is required for a clean diagnosis. Fortunately, detecting specific nucleotide sequences is a specialty for synthetic gene circuits.
RNA Detection With Toehold Switches
The working engines of the new system are RNA toehold switches. A toehold switch is a stable hairpin that hides the ribosome binding site (RBS) and the start codon in front of a reporter gene, preventing translation. However, a complementary “trigger” RNA, e.g., the ssRNA comprising the Zika genome, can bind the switch region, opening up the RBS and turning on the reporter. This study tested two new toehold switch designs, improving the signal-to-noise ratio.
Simple RNA Amplification And Extraction
The toehold switch circuits could sense Zika RNA down to 30 nM, but real-world samples would be more than 106 times lower. To amplify the sample RNA, the team adapted nucleic acid sequence-based amplification (NASBA). In this method, sample RNA is reverse transcribed into DNA containing a strong promoter, which directs transcription of that cDNA back into many more copies of RNA. To simplify their protocol and eliminate equipment cost, the team skipped a traditional 65C heating step, showing the reaction worked fine at a steady 41C.
Amplifying RNA only works if you have some RNA to start with, so the team showed that a simple 2 minute boiling step could release RNA from RNA viruses, including Zika.
Single-Base Resolution With CRISPR
Toehold switches are sequence-specific, but they don’t quite have single-base resolution, which is needed to distinguish between related strains. To improve their resolution, the team added CRISPR/Cas9 to the NASBA amplification. Using a sgRNA targeting the trigger RNA sequence, they took advantage of a PAM site present in only one strain of the virus. The cDNA from the strain with the PAM was cleaved, preventing NASBA amplification, while still allowing the second strain to be amplified and detected.
Freeze-Dried Circuits In A Box
If freeze-drying makes things better, all-in-one boxes are of course the only logical next step. To that end, the team developed a cheap, battery-powered box that can hold the circuit-soaked filter paper and read out its color change over time. At $250, this could enable cheap virus detection in the field.
This study focused on the currently zeitgeist-residing Zika virus, but on paper, their method should work equally well for any RNA virus. Not only does it offer cheap, robust, single-base pair genomic resolution, but designing and manufacturing new toehold switch circuits for any other viral sequence should be quick and easy.
For more info (and at least from our printer, better texture than freeze-dried ice cream) check out the paper in Cell, May 2016.