TWJ-Toehold


Overview

TWJ-Toehold is a variant of the SDA reaction that introduces the TWJ structure and was reported by Chen et al. 1.
For more information about the principle of TWJ and SDA, see Proposed Implementation_Amplification.
For more information about the actual experimental procedure of TWJ-Toehold, see Experiments_ TWJ-Toehold.

  1. Target DNA, template DNA, and helper DNA form a TWJ complex.
  2. Polymerase extends the 3' end of the helper DNA.
  3. Nickase introduces a nick.
  4. From the nick, the polymerase displaces the previous strand while continuing to extend the new strand.
  5. Steps 3-4 are repeated, leading to the production of large amounts of the strand on the 3' side of the helper DNA, referred to as "trigger DNA."
  6. The reaction then branches into two main pathways:
    1. Recycling of target and extended helper:
      1. Trigger DNA binds complementary to the 5' side of the template DNA.
      2. Polymerase displaces the extended helper DNA from the template while extending the 3' side of the trigger DNA.
      3. The target DNA, template DNA, and extended helper DNA form the TWJ complex.
      4. Steps i-iii are repeated, leading to the production of large amounts of dsDNA.
    2. SDA reaction by trigger and template:
      1. The trigger and template form a complex.
      2. Polymerase extends the 3' side of the trigger, producing large amounts of dsDNA.
      3. Nickase introduces a nick.
      4. Polymerase displaces the previous strand while extending the new one from the nick.
      5. Steps iii and iv are repeated, producing large amounts of the strand on the 3' side of the trigger DNA (trigger DNA itself).

TWJ-Toehold reaction mechanism:

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1. Preliminary Experiments

Purpose:

We attempted to confirm the amplification by the mechanism in the paper. In the paper, the amplified products (trigger) activate CRISPR-Cas14a for nonspecific collateral cleavage of ss DNA reporter, which turn on for fluorescence. In contrast, we measured fluorescence intensity using SYBR Green Ⅰ. Since TWJ-Toehold is very similar to ThisAmp in the reaction mechanism, we thought that we could perform the same experiment using SYBR Green Ⅰ with TWJ-Toehold as we did with ThisAmp.

Result:

The fluorescence changes were plotted below.

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Consideration:

LoD was 10 pM. In the paper, the fluorescence intensity is measured by connecting it to the CRISPR-Cas14a system, and the LoD at that time was 50 fM. We thought that we could further lower the LoD even if the reaction was not connected to the CRISPR system, so we decided to conduct tuning.

2. Tuning

Purpose:

Tuning of the helper concentration was performed to further lower the LoD.

Result:

The fluorescence changes were plotted below.

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Consideration:

The difference in the time at which the amplification curve starts rising when the target concentration is 1 pM and 0 M was the largest at the condition with the helper concentration 2.5 nM, as in the paper.

3. Re-Experiment

Purpose:

To confirm the LoD of this system, we performed the experiment again.

Result:

Fluorescence changes were obtained as shown in the graph below.

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Consideration:

The LoD was 10 pM.

Conclusion

TWJ-Toehold had the LoD of 10 pM. This reaction is useful in that it can form a TWJ and amplify without the need for temperature changes at a constant 55 ℃, and has a detection limit \(10^1\) orders lower than ThisAmp, but the target is 51 mer long, making it unsuitable for miRNA amplification. In addition, since the target, template, and helper must be annealed before the enzyme is added, it is difficult to connect other amplification mechanisms before this reaction in a one-pot amplification system. Furthermore, since the main amplification product is dsDNA, it would also be difficult to connect other amplification mechanisms behind this reaction.

References

  1. Chen, M., Jiang, X., Hu, Q., Long, J., He, J., Wu, Y., Wu, Z., Niu, Y., Jing, C., & Yang, X. (2024). Toehold-Containing Three-Way Junction-Initiated Multiple Exponential Amplification and CRISPR/Cas14a Assistant Magnetic Separation Enhanced Visual Detection of Mycobacterium tuberculosis. ACS Sensors, 9(1), 62-72. https://doi.org/10.1021/acssensors.3c01622