TWJ-2cycle is a variant of the SDA reaction that introduces the TWJ structure and was reported by Zhang et al. 1. This method is expected to have high specificity.
For more information about the principle of TWJ and SDA, see Proposed Implementation_Amplification.
The series of reactions proceed at an isothermal temperature of 37 ℃
First, we attempted to confirm the level of detection limit by conducting experiments based on the paper 1.
In the paper, Klenow (exo-) DNA Polymerase was used as the polymerase, but we used Bst LF, whose amplification at 37 ℃ has been confirmed in previous experiments.
The fluorescence changes were plotted below.
No amplification was observed at all.
The optimal conditions for Nickase and Polymerase are usually different. The optimal condition for Nt.BbvCI, the Nickase used in 1, is rCutSmart buffer, and NEBuffer 2.1 is the optimal condition for Bst LF, also used as Polymerase 2 3.
ThermoPol buffer, which is the optimal condition for Polymerase, was used based on the reference, but experience leading up to this experiment showed that using the buffer optimized for Nickase was a more successful finding.
Buffer was changed to rCutSmart Buffer, the optimal condition for Nt.BbvCI, and the experiment was performed.
The fluorescence changes were plotted below.
Amplification was confirmed by reaction in rCutSmart Buffer.
Even at a target concentration of 10 nM, it took about 100 min for the fluorescence intensity to increase. Amplification efficiency is insufficient for use as an amplification system for POIROT, which aims at detection and quantification at lower concentrations, and tuning or coupling with other methods should be considered.
Polymerase / Nickase was tuned for concentration.
The fluorescence changes were plotted below.
S/N ratio (ratio of fluorescence intensity to NC at target concentration of 1 nM) is an important factor in determining the quality of an amplification system. We determined the S/N for each condition at each time and its maximum value.
Polymerase 0.063 U/µL and Nickase 0.12 U/µL or Polymerase 0.063 U/µL and Nickase 0.16 U/µL showed good S/N ratios; the latter showed higher values than the former in S/N ratio, but the amplification curve shows no significant difference between them. Since enzymes are expensive and it is desirable to reduce the amount used, we considered 0.063 U/μL for Polymerase and 0.12 U/μL for Nickase to be optimal and decided to use these conditions for future experiments.
In many literatures, TWJ complexes are reported to be superior in terms of specificity. We have confirmed by experiment whether the formation of the TWJ complex has higher sequence specificity than hybridization of a pair of complementary DNAs. The results were fed back to Dry Lab and the reason why the TWJ complex is formed with high sequence specificity was discussed.
Similar sequences are known to exist in hsa-let-7b, the target miRNA of this system, and are named hsa-let-7a, hsa-let-7c, hsa-let-7d,..., hsa-let-7i. In the reference1, specificity has been studied for these series. We evaluated the specificity of the let-7 series plus an originally designed single nucleotide variant of let-7b.
The fluorescence changes were plotted below.
NJ: Sequence recognition using hybridization of complementary pairs of DNA
TWJ: Sequence recognition using the TWJ complex
In NJ-SDA, the amplification reaction occurred faster in some of the let-7 series than in NC, and in the single nucleotide mutant, the mutant was amplified faster than the perfect-match target.
In TWJ-SDA, the amplification curve is similar to that of Negative Control except for target in the let-7 series. This indicates that TWJ-SDA can ensure high specificity compared to NJ, that is, when the three-way complex is not formed.
The fluorescence intensity at 40 min after the start of the reaction, including the error range, is evaluated in the figure below (error bars indicate standard error).
For subsets of the let-7 series, no significant differences in fluorescence intensity were observed in TWJ compared to NC except for let-7b.
In NJ, let-7c was not significantly different from let-7b, and let-7e and let-7i showed significantly higher fluorescence intensity than NC.
Single nucleotide mutants showed no significant difference from the Negative Control except for mutants at 1 mer, 9 mer, and 11 mer from the 5' end.
Mutations from 5' end to 1 mer and 9 mer showed significantly higher fluorescence intensity after 40 min compared to NC, especially the mutation from 5' end to 1 mer, which was not significantly different from the perfect-match let-7b.
Thus, it was confirmed that the specificity changed depending on the position of the mutation.
For a discussion of the reasons for this, click here:
The use of TWJ showed extremely high specificity compared to NJ, indicating that TWJ-SDA is an extremely promising system to ensure the target specificity required for POIROT. However, the amplification efficiency of TWJ-SDA alone is considered insufficient for amplification of extremely low concentrations of miRNA in a few tens of minutes. Therefore, it is necessary to combine the system with other mechanisms to increase amplification efficiency.
Qing, Z., Feng, C.,Feng, X., Yongxi, Z., & Chunhai, F. (2014). Target-Triggered Three-Way Junction Structure and Polymerase/Nicking Enzyme Synergetic Isothermal Quadratic DNA Machine for Highly Specific, One-Step, and Rapid MicroRNA Detection at Attomolar Level. Anal. Chem. 2014, 86, 16, 8098-8105. https://doi.org/10.1021/ac501038r
New England Biolabs. (n.d.). Bst DNA polymerase, large fragment. https://www.neb.com/ja-jp/products/m0275-bst-dna-polymerase-large-fragment
New England Biolabs. (n.d.). Nt.BbvCI (nicking endonuclease). https://www.neb.com/ja-jp/products/r0632-ntbbvci