The Three-Way-Junction SDA (TWJ-SDA) is a mechanism that initiates SDA based on a three-way junction complex formed by a short helper and a long template hybridizing with the target single-stranded nucleic acid. Ying Xu et al. reported achieving high sensitivity by using a hairpin-structured template and employing the ssDNA (trigger) produced from TWJ-SDA as a starting point for amplification in EXPAR 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-EXPAR, see Experiments_TWJ-EXPAR.
This experiment aimed to confirm amplification from single-stranded nucleic acids based on the mechanisms described in the paper. Molecular beacon (MB) is used as the template for the latter half of amplification, EXPAR, in the paper. This choice is made for convenience in verifying amplification. In principle, it is equally acceptable to use unmodified ssDNA with the same sequence. Therefore, we conducted experiments using ssDNA with the identical sequence to the MB as the template and measured the fluorescence intensity of SYBR Green Ⅰ.
In the following sections, we denote the ssDNA that forms the three-way complex in the first stage as template1 and helper1, the ssDNA that forms the three-way complex in the second stage as template2 and helper2 and the ssDNA product from the first stage of amplification as trigger.
In the fluorescence measurements using both the MB and SYBR Green Ⅰ, no increase in fluorescence intensity dependent on target concentration was observed at all.
Amplification was not confirmed in either the SYBR Green Ⅰ measurements or the experiments using the MB as described in the paper. In the SYBR Green Ⅰ experiments, the concentration of the DNA used as the template was very high, and it is likely that these templates also possessed hairpin structures. Since SYBR Green Ⅰ primarily labels dsDNA, the background fluorescence intensity may have been high, potentially hindering accurate quantification of the amplification products.
However, the lack of amplification even with the MB as described in the paper suggests a need to segmentalize the mechanism further to identify bottlenecks, as well as to optimize the experimental conditions.
In the preliminary experiments using SYBR Green Ⅰ, the fluorescence intensity was significantly higher compared to the EXPAR conditions. According to reference 2, monitoring amplification reactions using SYBR Green Ⅰ is effective when the concentration of the DNA is extremely high or DNA capable of forming secondary structures do not exist in the system. In our setup, we were using template2 at a very high concentration of 1.3 µM, which was expected to have a hairpin structure that includes regions of dsDNA. Therefore, tracking the reaction via fluorescence intensity measurements with SYBR Green Ⅰ is likely inappropriate.
To address this, we investigated the fluorescence intensity as the concentrations of template2 and helper2 were gradually decreased.
No increase in fluorescence intensity was observed at any concentration. In all conditions, a final target concentration of 1 nM was added.
Regardless of the concentrations of template2 and helper2, the fluorescence intensity did not change. This suggests that a fundamental reconsideration of the amplification system is necessary.
We conducted the experiments in two phases, as illustrated in the diagram below.
To confirm whether amplification in the first phase occurs successfully.
We designed the mechanism as described above, added SYBR Green Ⅰ, and incubated at 37 ℃ to measure the fluorescence intensity.
The changes in fluorescence intensity are shown in the graph below.
When the target concentration was 1 µM, there was an initial high fluorescence intensity that subsequently decreased. The same trend was observed when using miRNA as the target, with very little fluorescence attenuation. Although a slight increase in fluorescence intensity was noted at concentrations below 100 nM, no amplification dependent on target concentration was observed.
In both cases, whether using miRNA or ssDNA as the target, amplification from the first phase could not be confirmed.
To confirm whether amplification in the second half of the reaction occurs successfully. We designed the mechanism as shown below and, in addition to fluorescence measurements using the MB, added SYBR Green Ⅰ with unmodified ssDNA that takes a hairpin structure as the template, incubating at 37 ℃ to measure the fluorescence intensity.
As shown in the graph below, no increase in fluorescence was observed when measuring the fluorescence intensity with SYBR Green Ⅰ.
When observed using the MB, an increase in fluorescence intensity over time was noted. However, this increase was not dependent on the concentration of the trigger.
In all experiments, it is unlikely that amplification of ssDNA occurred as we had expected. Both the first and second phases of the mechanism appear to have underlying problems.
In this mechanism, neither the first nor the second phase is functioning effectively, leading us to conclude that it is difficult to use POIROT as an amplification mechanism. Therefore, we considered alternative approaches.
Ying, X., Yu, W., Su, Liu., Jinghua, Y., Hongzhi, W., Yuna, G., & Jiadong, H. (2016).Ultrasensitive and rapid detection of miRNA with three-way junction structure-based trigger-assisted exponential enzymatic amplification. Biosensors and Bioelectronics, 81, 236-241. https://doi.org/10.1016/j.bios.2016.02.034
Wang, C., & Yang, C. J. (2013). Application of molecular beacons in real-time PCR. In M. D. Teintze (Ed.), Molecular Beacons (pp. 45-59). Springer. https://doi.org/10.1007/978-3-642-39109-5_3