Team:SJTU-BioX-Shanghai/Proof Of Concept
To demonstrate the actual potential and feasibility of our project concept, we further verified and implemented our hypothesis on the basis of several papers. We chose to use experimental and modeling approaches，using gardquimod aptazyme (Gard-337) and theophylline aptazyme (Theo-AAG/AAAAA) with or without target for our De novo Rapid in Vitro Evolution of RNA biosensors(DRIVER) and lateral flow assay (LFA) experiments, and adopting modeling tools to simulate our T7 transcription system and DRIVER system. These results provided preliminary proof that this technique can detect and report the presence of target, and demonstrate the potential of this technique to be further optimized into an adaptable point-of-care diagnostic tool.
The first proof-of-concept experiment we conducted was to verify the feasibility of using DRIVER technology to select the aptamers corresponding to our target groups. The verification process in this step was mainly carried out by testing whether the integrity of the library we constructed could be guaranteed after several rounds of selecting1. We ran transcription product of selection round 12 of DRIVER on 10% RNA urea denatured PAGE (Fig. 1). The verification results showed that the library constructed by us could exist stably after 12 rounds of DRIVER, which proved that DRIVER technology could select the aptazymes we need in the future.
Figure 1 T7 transcription product of selection round 12
Before our LFA approaches the practical application further, we must understand the relationship between the target concentration added in transcription experiment and the T line’s color development on the test paper through experiments, so as to ensure the sensitivity of the LFA to target of different concentrations. We chose to add target (gardiquimod) of different concentrations into the T7 transcription system for transcription experiments2, perform PAGE (Fig.2) and load the product on the LFA to observe (Fig.3a). Finally, we analyzed the intensity of T line’s color (Fig.3b). And when the concentration of gardiquimod reaches 10 micromoles per liter, there is an obvious difference from the case without target.
Figure 2 T7 transcription product with different concentrations of gardiquimod
Figure 3 T7 transcription product on LFA with different concentrations of gardiquimod(volume=2μl) in the 20μl transcription system
Our simulation process of T7 transcription system reaction please click there.
Compared with the transcription system, the modeling process of DRIVER system is more complicated3. In each round of our PCR in the experimental process, the template of aptazyme that does not self-cleave with the addition of ligand will be amplified, otherwise the template of self-cleaving aptazyme will not be amplified and will be further diluted in the following steps. The amplification multiple of each round of PCR is constrained by the number of reaction rounds. When ligand is added, DNA molecules will be attenuated due to irresistible factors in the reaction before PCR amplification and the attenuation rate is about 75%. In the simulation process, we use a general formula to express the result after each DRIVER. According to the formula, the change of the concentration of DNA in each round is not determined by whether to add ligand in each round, but only related to the number of reaction rounds. In order to make the number of rounds random, we randomly added ligand for 3-5 rounds at the beginning and did not add ligand for 3-5 rounds, with an amplification multiple of 4-8 times for the enriched aptazymes in each round. In the subsequent process, we randomly added ligand in 22-25 rounds, and other 25-28 rounds did not add the ligand, and the amplification multiple changed from 1 to 44,5. For the initial concentration of DNA with different shearing properties added to the library, we set it as shown in the figure below. Overall, the curve is high in the middle and low on both sides, and there is a cliff-like decline. The figure below is the result we got after selecting. After selecting, the amount of the part we want (the area in the lower right corner) is already very remarkable. Due to the high initial content, there is still a small amount of residue in the middle, but the significance is not high.
Figure 4 Cleavage ratio with/without ligand DNA before and after selecting.
The hardware body of our project is a smooth, hollow shell with T7 RNA Polymerase@ @50U/µl and RNase inhibitor required for the reaction, along with solutions for mixing and washing, strips of test strip for target detection, and adhesive bandage. According to statistical calculation, the total cost of our design system is 39.06 USD, of which 27.1 USD is used for transcription reaction, and the cost of test strip accounts for 14.46%. Future work will focus on implementing transcription-free hardware systems that dry RNA by ethanol precipitation on a loading pad and add RNA inhibitors and 2x quantitative START probes to PBS solutions to further shorten our assay time.
Our hardware cost estimation form please click here.
Figure 5 Hardware
Figure 6 Bin charts of system evaluation before and after improvement
