Designed sequences


Overview

In SDA using TWJ, amplification can be performed by changing the template and helper sequences to target any miRNA in principle. However, it has been suggested that the stability of the TWJ portion changes the specificity and sensitivity 1.

Designing appropriate template and helper sequences according to the miRNA sequence to establish desired amplification is essential for the versatility of POIROT. Amplification experiments with various sequences were performed to obtain parameters for sequence design by Dry Lab.

We designed a number of templates and helpers to amplify hsa-miR-10b-5p, hsa-miR-375, and hsa-miR-30d-5p (referred to as Biomarker 1, 2, and 3 in this order), which have been reported as biomarkers for glaucoma, and conducted experiments.
More information about biomaker miRNA, click here:

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Sequence Design Part.1

Purpose:

To begin experiments using our own sequence design, we first modified only the miRNA-complementary sequence of the template used in the TWJ-2cycle. The template and helper we used in the TWJ-2cycle recognize let-7b, however, by changing miRNA-complementary sequence, they then recognize other miRNA.

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We then changed the hybridization length of the template and helper to 5-7 bp.

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We then measured the changes in fluorescence intensity under various target concentrations.

The experiments were performed using SYBR Green Ⅰ under the conditions tuned in TWJ-2cycle for the type and concentration of polymerase and nickase.

In the following, the helper for Biomarker n, which hybridizes with template and m bp, is referred to as helper n-m.

Result:

For each biomarker, the following measurements were obtained.

Biomarker1

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Biomarker2

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Biomarker3

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Consideration:

Amplification was confirmed for all helpers. In the case of all biomarker targets, the longer the chain length of hybridization between template and helper, the larger the initial slope of the amplification curve tends to be. To sum it up, the longer the hybridization length of the template and helper was, the greater the amplification rate became.

As for the difference in the fluorescence curve in response to the target concentration, we were able to clearly distinguish between NC and 1 pM or higher for biomarker1 when helper1-7 was used. Furthermore, looking at the slope in the first linear amplification section, it was confirmed that there was a positive correlation with target concentration in the region of 1 pM - 100 pM target concentration. In other words, the amplification rate of TWJ-SDA is considered to be determined by the target concentration.

However, for biomarker 2 and 3, there was no helper that showed a clear target concentration-dependent amplification rate.

The stability of the TWJ complex is related not only to the hybridization region between template and helper, but also to the region where the target and template hybridize. It will be necessary to conduct experiments by changing the hybridization length of the template and target to find the optimal TWJ structure.

Sequence Design Part.2

Plan:

As mentioned above, the stability of the TWJ complex is related not only to the hybridization region between template and helper, but also to the region where the target and template hybridize. For this reason, Dry Lab designed three types of templates for each biomarker, and the hybridize lengths of target and template, and helper and template were changed for further experiments independently.

For biomarker 1, 2, and 3, the hybridize lengths of template and target were 10, 11, and 12 bp, and the hybridize lengths of helper and template were 5, 6, 7, and 8 bp, respectively.

Result:

The following amplification curves were obtained. For each template/helper pair, the amplification curves are shown with (solid line) and without (dashdot line) the addition of the final 10 nM of target.

In the following, if the hybridize length of template and target is 10 bp, the hybridize length of helper and template is 5 bp, for instance, this experiment condition will be referred to as 10-5.

Biomarker1

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Biomarker2

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Biomarker3

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Consideration:

The ratio of the fluorescence intensity at the 10 nM (target) to that at 0 M after 30, 45, and 60 min was calculated as shown in the table below. Here, the ratio of the fluorescence intensity at each time minus the fluorescence intensity at the first cycle was calculated to exclude what was considered to be background fluorescence.

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The larger this ratio, the more clearly the target concentration can be distinguished.For Biomarker 1, when the condition was 12-5, that is, when the hybridize length between template and target was 12 bp, and the hybridize length between template and helper was 5 bp, the ratio was the largest.

Similarly, it was concluded that 10-5 or 12-7 for Biomarker2 and 12-6 for Biomarker3 would be optimal.
For more detailed analysis, click here:

References


  1. 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