Connection of Amplification and Cas


Overview

The experiments conducted so far aimed to establish the amplification system, allowing for the amplification of DNA in a manner dependent on the initial concentration of fM order biomarker miRNA by the selection of methods and tuning the concentrations of reagents. To visually represent the concentration of nucleic acids as an input signal, we aimed to use dsDNA as the final product and employ the collateral activity of Cas3 to quantify the biomarker miRNA.

1. Preliminary Experiments

Purpose:

To start, we performed a series of amplification reactions using biomarker 1, and then designed an elongation reaction for dsDNA using the final products of the multistep-SDA as primers. Similar to the Wet Results_CRISPR-Cas, we measured the collateral activity of Cas using an FQ probe.
For comparison, we designed an elongation reaction for dsDNA using the products of the 2-step multistep-SDA as the starting point, and conducted experiments on this as well, utilizing an FQ probe in the same manner.

Result:

The fluorescence changes were plotted below.

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Consideration:

In both cases, an increase in fluorescence intensity was observed at all target concentrations, including the NC. However, no differences based on target concentration were noted, making it impossible to distinguish between the NC and other samples. From the experimental results conducted by varying dsDNA concentrations, it was evident that the collateral activity of Cas3 is completely dependent on the concentration of the activated dsDNA. This suggests that the final product dsDNA is produced in a independent manner relative to the target in the amplification system.

2. Consideration of Two-pots Reaction

Purpose:

The experiment of 1. was conducted in a one-pot manner, meaning that amplification and the cleavage of ssDNA by Cas3's collateral activity occurred simultaneously. This could lead to different behavior, as the template may also be cleaved, compared to previous experiments that focused solely on amplification.
To address this, we performed the amplification for a fixed incubation time (in this case, 40 minutes) before adding Cas3 protein, cascade protein, and the FQ probe, followed by measuring changes in fluorescence intensity. This approach allows the amplification reaction to proceed without the influence of Cas3's collateral activity, leading to collateral activity dependent on the concentration of the dsDNA product generated from amplification.
Therefore, we expect to observe rapid changes corresponding to the initial concentration.

Result:

The fluorescence changes were plotted below.

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Consideration:

Even in the two-pot reaction, amplification of the NC was observed. During the initial few minutes of fluorescence measurement, the slope for the NC was nearly indistinguishable from that of the target concentrations ranging from 1 fM to 10 pM. From the results of CRISPR-Cas, the slope of the graph in this section, that is, the cleavage rate due to collateral activity, should depend on the concentration of the produced dsDNA. Therefore, it suggests that there may be some problem in the amplification stage, leading to the final product dsDNA being produced in a target-independent manner.
To address this problem, we first conducted experiments by segmenting the mechanism to investigate which part of the amplification system was acting as a bottleneck.

3. n-step-SDA→Cas3

Purpose:

To identify the bottleneck, we designed a system that connects the ds-template after the 0, 1, 2, and 3 steps of SDA and measured the collateral activity of Cas3. In each experiment, the target concentration was varied around the expected amount of intermediate products when starting with 1 fM of miRNA.

Result:

The fluorescence changes were plotted below.

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Consideration:

In the case of the dsDNA extension reaction alone, no increase in NC fluorescence intensity was observed, and the graph showed a slope dependent on target concentration. When connecting the 1step-SDA with the dsDNA extension reaction, the final concentration displayed a slope dependent on the target concentration in the range of 0 - 10 nM. However, an increase in NC fluorescence intensity was observed.
When the 2step-SDA was connected with the dsDNA extension reaction, the target final concentration could be clearly distinguished between 0 M and above 1 nM, but differences above 1 nM were not distinguishable. In the case of connecting the 3step-SDA with the dsDNA extension reaction, the fluorescence intensity in NC increased at a rate similar to other conditions, making it impossible to differentiate between conditions.

From these results, it can be concluded that while the dsDNA extension reaction alone allows for quantitative detection while suppressing NC, the increase in fluorescence intensity in NC when connected with SDA indicates that tuning the template concentration is necessary.

4. Tuning of Template Concentration

Purpose:

When connecting the 1step-SDA with the dsDNA extension reaction, we need to find the appropriate ds-template concentration.
In condition Establishment of Amplification, the concentration of template 3 is 40 nM, and the concentration ratio between templates is tuned to 2, resulting in a ds-template concentration of 80 nM. However, to suppress NC while allowing for rapid amplification in the presence of the target, experiments were conducted by varying the concentration.

>Result:

The fluorescence changes were plotted below.

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Consideration:

As the concentration of the ds-template increased, a trend was observed where the amplification of the NC occurred more rapidly. It was determined that a final concentration of either 8 nM or 16 nM of ds-template would be promising, as it allows for clear differentiation between low target concentrations and the NC while effectively suppressing the NC.
Therefore, we tuned the ds-template concentration to 12 nM and conducted experiments connected through a 3step-SDA.

5. 3step-SDA > ds > Cas3: Under Tuned Conditions

Purpose:

We tuned the ds-template concentration to 12 nM and conducted experiments connected through a 3step-SDA. For comparison, we also performed experiments at the original concentration of 80 nM.

Result:

The fluorescence changes were plotted below.

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Consideration:

The increase in fluorescence intensity at 12 nM of ds-template was also observed at NCs, but 1 fM and NCs were clearly distinguishable.
In the control experiment, when the ds-template concentration was 80 nM, the fluorescence intensities at all concentrations showed similar changes and could not be used as a detection mechanism.
Tuning made it possible to link 3step-SDA and cas3 to detect nucleic acids at low concentrations.

Conclusion

We succeeded in detecting 1 fM nucleic acids by our mechanism of linking 3step-SDA and ds-Amplification by tuning the template concentration. The mechanism linking miRNA to Cas3 via TWJ has not been realized due to time constraints; however, it has been demonstrated that our mechanism can be used to detect nucleic acids at low concentrations using Cas3.
We were able to establish the mechanism of POIROT from Amplification to Detection by our previous experiments.
All previous experiments have measured fluorescence intensity by using a special machine. Finally, we aim at visual detection and quantification by using LFA.