Proof of Concept

The concentration of miRNA in tear fluid is extremely low. Total concentration is estimated to be in the hundreds of fM ¹. Given that, POIROT needs to detect target miRNAs at approximately 1 fM with amplification. We designed a method that connects TWJ-SDA ² with Multistep-SDA ³. No experiments connecting these methods have been reported so far.

These amplification systems are original, necessitating the development of a new model. Dry Lab conducted fitting the experimental data, reproduced the experimental results. By doing so, Dry Lab aimed to optimize template concentration. This model is by no means an armchair philosophy.

We then proceeded with amplification. In previous research by Yoshimi et al. ⁴, nucleic acid amplification was performed in a two-pot reaction using the LAMP before mixing the Cas3 solution. We combined the amplification system with the Cas3 system and successfully developed a detection system. We constructed a one-pot system. One-pot reaction is more user-friendly because no operation is needed during reaction.

By connecting TWJ-SDA and Multistep-SDA, we successfully amplified biomarker miRNA in artificial tear fluid with the concentration-dependent at the fM order.

Furthermore, we combined the amplification system with the Cas3 system, achieving one-pot nucleic acid detection. By connecting 3step-SDA and the Cas3 system using a uniquely developed sequence, we successfully detected nucleic acids at 1 fM.
For more detail, see Wet_Results.

Additionally, we conducted fitting to establish a model based on experimental data. This allowed us to verify various combinations of amplification methods in silico. By doing so, we demonstrated that the connection of TWJ-SDA > 3step-SDA is optimal for amplification. Furthermore, we were able to propose the optimal template concentration ratio to develop amplification efficiency.
For more detail, click here:

Dry Lab developed software to profile miRNAs in tear fluid and developed similar sequences to the input miRNA. Using this software, we found that tear fluid would contain miRNAs with one or two nucleotide substitutions from the biomarker miRNA.
For more detail, see Software.

Thus, POIROT must distinguish between similar sequences and amplify only the target miRNA. Wet Lab conducted experiments using subsets of target miRNAs and Single Nucleotide Polymorphisms (SNPs) with substitutions at various positions. Additionally, Dry Lab used these results to build a model for TWJ-SDA, aiming to provide a theoretical basis for the specificity based on the position of mutations in the miRNA.

As shown in the figure below, we measured the amplification curves by comparing TWJ and Non-Junction (NJ), using subsets and SNPs as targets.

Results were plotted below.

We proved that the formation of the TWJ complex enables high specificity. In particular, for the let-7 series, which are subsets of let-7b, no statistically significant difference was observed compared to the negative control. For more details, see Wet_Results.

Dry Lab attempted to reproduce these results and concluded that the high specificity of TWJ lies in the delicate thermodynamic stability of the TWJ complex. Based on this model analysis, it became possible to design template and helper sequences for even more target-specific amplification by forming an appropriately thermodynamically stable tripartite complex.

For more detail, click here.

POIROT is a POC device. Therefore, in order to ensure accurate results despite minor environmental fluctuations, a robust amplification mechanism is required. During the process of determining the amplification mechanism, the Dry Lab evaluated the robustness of various amplification methods and explored a better one that could maintain both robustness and specificity.
Also, Dry lab evaluated the robustness of Cas3's activity using Molecular Dynamics (MD) Simulation.

Through investigations of prior research ⁵, experimental results from Wet Lab, simulation outcomes from Dry Lab, it was shown that two amplification systems, TWJ-SDA > EXPAR and TWJ-SDA > 3step-SDA, provided sufficient amplification efficiency and a high positive-to-negative ratio (P/N ratio) for this project. Consequently, these two amplification systems were proposed as the final candidates. ODE-based simulations were conducted to compare the two systems' robustness to time and enzyme.
For time robustness, the change in amplification efficiency and the positive-to-negative ratio was analyzed as the reaction time varied. For enzyme robustness, the impact of varying enzyme activity was examined. The results demonstrated that in all six indicators (2x3), TWJ-SDA > 3step-SDA showed superior robustness. Therefore, TWJ-SDA > 3step-SDA was proposed as the amplification system to be used in the project.
For more detail, see Model_Specificity.
Using the Molecular Dynamics (MD) simulation on the Cascade-crRNA-DNA complex and the Cas12a-crRNA-DNA complex, Dry Lab evaluated the stability between CRISPR-Cas and dsDNA, final product of amplification. For more details, see MD Simulation.

The potential applications of POIROT are not limited to detecting and quantifying biomarker miRNAs for glaucoma. By combining miRNA profiling software with a theoretical understanding of the specificity of recognition within the TWJ complex, we can design appropriate templates and helpers for a comprehensive miRNAs.
Furthermore, the template sequences used to connect multistep-SDA and Cas3 can be universally applied. These template sequences have been registered in the Parts for future iGEMer.
Also, we developed a Software to propose a proper template and sequence for various miRNA. To see about our Software, click here;

To demonstrate the potential for sequence design, we designed sequences targeting three miRNAs that have been reported as biomarkers for glaucoma. We selected hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-30d-5p (referred to as Biomarker 1, 2, and 3, respectively) as targets. For each of these biomarker miRNAs, we performed TWJ-SDA and measured the fluorescence intensity corresponding to each biomarker.

For each biomarker, we calculated the S/N ratio (fluorescence with target / without target). Here, we present only the experimental results for Biomarker 2. For more detail, see Wet_Results.

From these results, we were able to experimentally identify appropriate templates and helpers that conduct sequence-dependent amplification for the three miRNAs we used. Using this experimental data, Dry Lab improved the ODE model and developed software to assist in designing optimal template and helper sequences for TWJ.
For more detail, click here:

Preliminary experiments conducted by the Wet Lab revealed that even when using commercially available strips, improper tuning can lead to false positives or, conversely, negative results under conditions where a positive outcome should be observed.

Wet lab assembled the LFA with reference to CONAN developed by Yoshimi et al. ⁴.The strip used was pre-installed with AuNPs with rb anti FITC antibody on the conjugate pad, streptavidin on the control line (C-line), and anti rb antibody on the test line (T-line). FITC-ssDNA-Biotin was used as a reporter ⁶. When CRISPR-Cas3 is activated, the ssDNA within the reporter molecule is cleaved, producing FITC-labeled reporter fragments and biotin-labeled reporter fragments. Using this, both the T-line and C-line turn red if the test is positive, and only the C-line turns red if the test is negative.