The amplification system used in this year's project can be divided into two parts. The first half is the TWJ-SDA reaction, and the second half is from the Multistep-SDA reaction to the cleavage of ssDNA by the Cas3-Cascade/crRNA complex. The ssDNA used in the first half consists of sequences that depend on the sequence of the biomarker to be detected, i.e., the miRNA to be recognized. On the other hand, the second half is composed of independent and robust sequences that are independent of the sequence of the target miRNA.
First, we will introduce the four template sequences used in the second half of the system as fundamental components. These parts are critical to establishing the foundation of POIROT's applicability and contribute to a system that significantly enhances amplification efficiency. We also introduce the primer, the product of each amplification step. These primers contribute to the modularity of the system.
| Name | Role | Description |
|---|---|---|
| BBa_K5232000 | template1 | The 3' end side has a sequence complementary to primer0 amplified by the TWJ-SDA reaction, and the 5' end side is the template strand for primer1. |
| BBa_K5232001 | template2 | The 3' end strand has a complementary sequence to primer1, and the 5' end strand is the template strand for primer2. |
| BBa_K5232002 | template3 | The 3' end strand has the complementary sequence to primer2, and the 5' end strand is the template strand for primer3. |
| BBa_K5232003 | ds-template | It has a sequence that is fully complementary to primer3, with the middle portion having a recognition sequence for the Cas3-Cascade/crRNA complex. |
| BBa_K5232004 | primer0 | The amplification product of TWJ-SDA, which is the sequence connecting the first and second halves. |
| BBa_K5232005 | primer1 | The amplification product of the first step of the Multistep-SDA reaction. |
| BBa_K5232006 | primer2 | The amplification product of the second step of the Multistep-SDA reaction. |
| BBa_K5232007 | primer3 | The product of the third amplification step of the Multistep-SDA reaction. |
Next, we introduced the parts of the first half. Although the sequences of template and helper that form the three way junction complex differ depending on the sequence of the target miRNA. Here we list template0 and helpers. Each part corresponds to miR-10b-5p, miR-375-5p, miR-30b-5p.
| Name | Role | Description |
|---|---|---|
| BBa_K5232008 | template0_10b-5p | The 3' end side has a sequence complementary to miR-10b-5p, and the 5' end side is the template strand for primer0. |
| BBa_K5232009 | helper_10b-5p | It has a sequence complementary to miR-10b-5p on the 5' end side and complementary to template0 on the 3' end side. |
| BBa_K5232010 | template0_375-5p | The 3' end side has a sequence complementary to miR-375-5p, and the 5' end side is the template strand for primer0. |
| BBa_K5232011 | helper_375-5p | It has a sequence complementary to miR-375-5p on the 5' end side and a sequence complementary to template0 on the 3' end side. |
| BBa_K5232012 | template0_30b-5p | The 3' end side has a sequence complementary to miR-30b-5p, and the 5' end side is the template strand of primer0. |
| BBa_K5232013 | helper_30b-5p | It has a sequence complementary to miR-30b-5p on the 5' end side and a sequence complementary to template0 on the 3' end side. |
Finally, the Cas3-Cascade/crRNA complex, which is activated by recognizing the elongation reaction product from ds-template(BBa_K5232003) and primer3(BBa_K5232007). For more information, see Proposed Implementation.
| Name | Description |
|---|---|
| BBa_K5232016 | Activated by recognizing the elongation products from ds-template (BBa_K5232003) and primer3 (BBa_K5232007). It is responsible for cleavage of ssDNA in the Reporter molecule by its collateral activity. Here we register crRNA. |