Our protocol describes the design, assembly, and validation of gene circuit-based diagnostics. These paper-based molecular diagnostics are low cost and sensitive, being able to detect clinically relevant concentrations of nucleic acids and can be designed to detect virtually any sequence. This is a platform technology that can be designed by users for their needs and have the potential to bring clinical-grade diagnostics into more decentralized dental resource settings.
Cell-free toehold switch sensor can be designed for virtually any nucleic acid-based target. Recent work has demonstrated cell-free diagnostics for Ebola, norovirus, SARS-CoV-2, C.difficile, and typhoid-causing bacteria. Demonstrating the procedure will be Severino Jefferson Rivero da Silva, a post-doctoral fellow, and Pouriya Bayat, a PhD student from my laboratory.
To design the toehold switch, identify the target sequences from the Zika virus genome and choose the Zika virus target sequences from the amplicons as described in the text protocol. Download the toehold switch design software package. Open MATLAB and navigate to the design software folder.
Input the target sequences into the design input file csv file located in the input subfolder. Select the parameters to use for the design function. Run the design function to generate the toehold switch designs for the targets of interest.
Upon completion, navigate to the final_designs folder and locate the top toehold switch design sequences and the corresponding target sequences in csv format spreadsheets. Ensure that the toehold switch DNA sequences generated by the algorithm contain the T7 promoter sequences at the five prime end and a conserved 21 nucleotide linker sequence at the three prime end. Use NCBI BLAST to screen the top toehold switch design sequences against other common viruses by checking for sequence homology.
Accept sequences with less than 40%hemology. Prepare solutions of the toehold switch hairpin DNA oligos and reverse amplification primers at a concentration of 10 micromolar in nuclease-free water. Assemble reactions in PCR tubes on ice according to this table.
Place the reaction tubes in a thermocycler, following the cycling conditions listed in this table. Analyze the PCR products on an agarose gel. Purify the PCR products and elute the DNA in a minimal volume of nuclease-free water to ensure a sufficiently high concentration.
Quantify the DNA using a spectrophotometer. Prepare a CPRG stock solution by dissolving 25 milligrams of the powder in one milliliter of nuclease-free water. Prepare a master mix on ice according to the standard protocol shown here.
Dispense the cell-free master mix into PCR tubes. For cell-free controls and switch alone controls, add nuclease-free water to a volume of 5.94 microliters. And for the reaction, add PCR purified toehold switch DNA to get a final concentration of 33 nanomolar.
To test the toehold switch and target RNA combination, add in vitro transcribed target RNA to a final concentration of one micromolar. Mix all reactions thoroughly by pipetting and centrifuge briefly. On a black clear bottomed 384-well plate, add 30 microliters of nuclease-free water to the wells surrounding the reaction wells.
Then using a two millimeter biopsy punch and tweezers, cut BSA blocked filter paper discs and place them in the reaction wells. Dispense 1.8 microliters from each reaction tube onto the filter paper discs in the 384-well plate in triplicate. Cover the plate with clear PCR film and place it in a plate reader.
Measure the absorbance at 570 nanometers at 37 degrees Celsius every minute for 130 minutes. Prepare a 25 micromolar stock solution of all forward and reverse primer sets in nuclease-free water. Set up a five microliter reaction using the master mix shown here.
Mix by pipetting until the white precipitate solubilizes and then dispense into PCR tubes. Add the forward and reverse primers to the appropriate tubes, followed by adding one microliter of either nuclease-free water or two picomolar of target trigger RNA. Mix by gentle pipetting and briefly spin down the tubes.
Set up the incubation protocol on a thermocycler as shown here. After 12 minutes, remove the tubes and add 1.25 microliters of the enzyme mix to each tube, followed by mixing and centrifugation. Return the tubes to the thermocycler after skipping the 41 degree Celsius hold step to start the one-hour reaction incubation.
Then to assess primer performance, assemble the paper-based cell-free toehold switch reactions and analyze the data. For sensitivity analysis, identify candidate primer pairs and prepare serial dilutions of target RNA in nuclease-free water. Repeat the NASBA and cell-free reactions with the selected primer sets in biological triplicates.
Using one microliter of extracted patient RNA, perform the NASBA amplification and paper-based cell-free reactions as shown in section five. Following the reactions, prepare the 384-well reaction plate and run the assay in a portable plate reader at 37 degrees Celsius. Then assemble the RT-qPCR components and add the reagents to a 1.5 milliliter microcentrifuge tube according to this table.
Mix the reaction by pipetting. Dispense 6.5 microliters into each well of a 96-well or 384-well PCR plate, followed by 3.5 microliters of each RNA template in triplicate. Place a PCR film over the top of the plate.
Centrifuge the 384-well plate at 600 times G for two minutes. Place the plate in an RT-qPCR machine and run the cycling conditions as shown here. Following the computational design, three toehold switches were constructed and analyzed using agarose gel electrophoresis.
A band around 3, 000 base pairs indicated a successful reaction. The toehold switches were assessed against their respective in vitro transcribed trigger RNA. While all three sensors displayed an increase in absorbance, sensor 27B had the most rapid on rate, whereas switches 33B and 47B had a lower on/off ratio indicating background activity and reduced specificity.
The fold change of absorbance at 570 nanometers showed that switch 27B has the best performance with an on/off signal ratio. Moreover, when coupled with NASBA, it can detect RNA at concentrations as low as 124 molecules per microliter indicating high sensitivity. Zika virus patient samples from Brazil were tested to assess the clinical diagnostic accuracy of the sensors using a portable plate reader.
A color change from yellow to purple identified a positive sample. The colorimetric response for each paper-based reaction was plotted over time by the integrated software on the PLUM portable plate reader. The samples that exceeded the threshold were considered positive.
The clinical performance of the sensor was established by comparison to RT-qPCR. The samples were considered positive when the cycle threshold value was equal to or less than 38. It is important to ensure that the results from toehold switch screens and NASBA primer sensitivity are reproducible and optimized before moving forward with patient trials.
Given its simplicity and adaptability, the diagnostic platform described here has paved the way for the development of new point of care tools that can bring benefits to the health system, especially for low-income countries.
