Results

HCR

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Overview

Hybridization Chain Reaction (HCR) is a method to amplify double-stranded DNA containing many nicks in an enzyme- free manner using microRNA or single-stranded DNA as input. It requires three different templates with a hairpin structure and reacts at 37 ℃. We performed experiments based on existing methods ¹.
For more information about the principle of HCR, see Proposed Implementation_Amplification
For more information about the actual experimental procedure of HCR, see Wet Experiments_HCR

1. Agarose Electrophoresis

We attempted to observe the amplified product by agarose gel electrophoresis.

Agarose gel electrophoresis showed low resolution and blurred peaks.

2. Capillary Electrophoresis

Because the peak was blurred in 1., we attempted to observe the amplified product using a more sophisticated capillary electrophoresis system. The concentration of target miRNA or ssDNA corresponding to that was used as the variable.

The elongated peaks were observed only at 1 µM for miRNA and at 1 µM and 100 nM for ssDNA. The paper reported that the dynamic detection range of CRISPR-HCR is 1 fM-100 nM and that of HCR is 10 pM-100 nM, but this could not be reproduced at all.

3. Optimization

Because the expected LoD could not be achieved in 2., changes were made to the experimental conditions.

• Annealing may make the hairpin structure too stable → Changed whether annealing was performed or not in the experiment
• The incubation time may not be long enough → Changed the incubation time
• The reaction between miRNA and H1 may be too slow in the first step → Changed the H1 concentration

• Without annealing, the amplified product was confirmed even in the negative control.On the other hand, without annealing, the amplified product was obviously detected around 52 bp. Therefore, annealing operation was thought to be essential.

• The results were similar for all incubation times after 10 min, indicating that an incubation time of 10 min is sufficient when the target ssDNA is 1 μM.

• The H1 concentration → When peak was observed, there was a tendency for lower H1 concentrations to produce fewer amplification products. For more detailed study, the concentration corresponding to Cas target was calculated from the molecular weight and concentration values of the peak.

The paper connects the products of HCR to Cas. Therefore, the concentration of target ssDNA/miRNA and the concentration of Cas target in the amplified product are expected to be correlated. It is expected that long amplified products contain more Cas targets and short amplified products contain fewer Cas targets.

Since one Cas target is contained per H2 or H3, one Cas target is contained per 60 bp of amplified product. 1 bp is approximately 330 Da, so this formula can be used to calculate the Cas target concentrations, taking into account all Cas targets contained in the various peaks.

4. Peak Attribution

For the results in 2., the peaks obtained by capillary electrophoresis were attributed to the assumed amplification product.

Capillary electrophoresis outputs the “molecular weight" and “concentration" of the peaks.Of these, the “molecular weight" was calculated by applying the molecular weight of the peak to the width of the upper and lower ladder of known molecular weight. If the peak is dsDNA, this calculation method can be used, but the amplified product of HCR is something like dsDNA with many nicks, and a discrepancy between the “molecular weight" calculated by the machine and the actual molecular weight is expected to occur.

Therefore, we avoided this problem by applying an amplified product with a value close to the “molecular weight" of the peak during attribution. Fortunately, the attribution was not difficult because, in principle, there are only a few amplification products with molecular weights close to the molecular weight of the HCR.

The results of the attribution showed that the amplification products are oligomers of n=10 at the longest. This indicates that the amplification efficiency of HCR is inadequate. The MultiNA we used has a lower detection limit of 0.2 ng/µL for DNA and a quantification range of 0.5-50 ng/µL ². Therefore, the lack of detection when the target was at low concentrations could be due to the fact that it was below the detection limit of MultiNA. However, based on the attribution of this peak, only n=10 oligomers at most were produced, and it would be difficult to amplify a concentration of PAM sufficient to activate Cas from a target of a few fM.

HCR

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1. Jia, H.-Y., Zhao, H.-L., Wang, T., Chen, P.-R., Yin, B.-C., & Ye, B.-C. (2022). A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction. Biosensors and Bioelectronics, 211, 114382.https://doi.org/https://doi.org/10.1016/j.bios.2022.114382

2. MultiNA https://www.an.shimadzu.co.jp/sites/an.shimadzu.co.jp/files/pim/pim_document_file/an_jp/brochures/20686/c297-0450.pdf

EXPAR

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Overview

Exponential amplification reaction (EXPAR) is a reaction in which SDA occurs exponentially. After receiving miRNA or ssDNA as input and hybridizing them with template ssDNA, DNA polymerase with strand displacement activity extends to the 5' end of the template to form dsDNA. The restriction enzyme recognition site on the dsDNA is nicked by nickase, and the polymerase recruits to the nicking site to cause strand displacement amplification. If the sequence of ssDNA of the amplified product is the same as the sequence of the input signal, the amplified product plays the same role as the target, and exponential amplification occurs. A 50 ℃ reaction is possible by using Bst 2.0 and Nt.BstNBI. We experimented with existing methods ¹.
For more information about the principle of SDA, see Proposed Implementation_Amplification.
For more information about the actual experimental procedure of EXPAR, see Experiments_EXPAR.

1. Preliminary Experiment

We attempted to confirm the amplification in the mechanism of the paper. The paper used dsGreen, but we used SYBR Green Ⅰ. Since SYBR Green Ⅰ is a double-stranded DNA staining reagent with the same performance as dsGreen ², this change does not affect the experimental results. In addition, OPC-purified oligo DNA was used.

The fluorescence changes were plotted below.

As in the paper, we were able to distinguish between the amplification curves for target 1 pM and 0 M. The amplification curve for target 1 pM started to rise about 7 min after the start of the reaction, which is consistent with the paper.

2. Heat Inactivation of Nickase

The reaction temperature of EXPAR is 50 ℃. In designing the ODE model of EXPAR in the Dry Lab, it was necessary to consider thermal inactivation of nickase during the reaction. Therefore, we conducted an experiment to confirm the thermal inactivation of nickase in the Wet Lab. Using the protocol in the ODE model paper ³ as a reference, we performed amplification reactions as usual and measured changes in fluorescence intensity using pre-incubated nickase solution for a certain period of time. The conditions other than pre-incubation of the nickase were the same as in the preliminary experiment.
For more information about the ODE model, see Model

The fluorescence changes were plotted below. Nickase was not added in NC.

No change in the amplification curve due to the pre-incubation time of the nickase was observed. It can be considered that changes in the enzymatic activity of the nickase due to reaction time are negligible.

/a>3. Comparison of Purification Level

We purchased OPC-purified oligo DNA and PAGE-purified oligo DNA. We performed an experiment to see if the difference in amplification efficiency between these two purification methods makes a difference.

The left graph shows the result with OPC-purified oligo DNA and the right graph shows the result with PAGE-purified oligo DNA.

The fluorescence intensity was higher when PAGE-purified oligo DNA was used; since PAGE purification is a method that yields highly purified oligo DNA ⁴, the higher fluorescence intensity could be attributed to the higher concentration of the oligo DNA of interest in the same volume of solution. As for the amplification speed, there was almost no difference depending on the purification method.

When OPC-purified oligo DNA was used, the experimental procedure was the same as in the 1. Preliminary Experiment, but the amplification speed was about five times faster than in the 1.

The left graph shows the fluorescence change in 1. and the right graph shows the fluorescence change when OPC-purified oligo DNA was used in 3. After rearranging the conditions, it was found that Bst 2.0, a polymerase, was purchased again between section 2. and 3., and that only the lot of Bst 2.0 was different between section 1. and 3. Therefore, we concluded that the difference in amplification speed between the two was due to the difference in Bst 2.0 lot. In subsequent experiments, we used the new Bst 2.0 lot and re-examine the response to template / nickase / polymerace concentration.

4. Tuning of Template Concentration

An attempt was made to further lower the LoD by tuning the template concentration.

The fluorescence changes were plotted below.

The higher the template concentration, the stronger the fluorescence intensity. The overall amplification speed was so fast that differences in amplification speed due to differences in template concentration were buried, and differences in amplification speed could not be measured. Since lowering the template concentration decreases the amount of amplified product, it is desirable not to lower the template concentration for the purpose of amplifying and quantifying nucleic acids. Therefore, in subsequent experiments, template concentration was set at 25 nM as in the paper.

5. Tuning of Nickase Concentration

Attempts were made to further lower the LoD by tuning the nickase concentration.

The largest difference in the time for the amplification curve to rise between the target concentration of 100 pM and 0 M was observed when the nickase concentration was 1/2 times of that in the paper (0.125 U/µL). We considered the possibility of increasing the difference in the time until the amplification curve rises between 100 pM and 0 M by further decreasing the nickase concentration.

6. Tuning of Polymerase Concentration

We attempted to further lower the LoD by tuning the polymerase concentration. Nickase concentration was set to 1/2 of the published value (0.125 U/µL).

The fluorescence changes were plotted below.

The difference in the time to amplification curve between target concentration 100 pM and 0 M was greatest when the polymerase concentration was 1/4 of the published value (0.02 U/µL). We considered the possibility of increasing the difference in the time to amplification curve rise between the 100 pM and 0 M by further lowering the polymerase concentration.

7. Tuning of Polymerase and Nickase Concentration

We attempted to further lower the LoD by simultaneously tuning the polymerase and nickase concentrations.

Fluorescence changes were obtained as shown in the graph below.

When the polymerase concentration was 1/16 times of that in the paper (0.005 U/µL) and the nickase concentration was 1/8 times of that in the paper (0.0625 U/µL), the amplification curves at 1 pM and 0 M could be distinguished.

8. Re-experiment under Tuned Conditions

To observe the LoD of this system, the experiment was repeated with a lower target concentration using the conditioning obtained in section 7.

The fluorescence changes were plotted below.

LoD was 1 pM.

Conclusion

The LoD was 1 pM when the new lot of Bst 2.0 was used with a polymerase concentration of 0.005 U/µL and nickase concentration of 0.0625 U/µL. EXPAR is an efficient amplification system and is promising to achieve the required amplification efficiency for POIROT within 30 min. On the other hand, EXPAR was shown to be unstable and highly dependent on the lot of the enzyme. The inclusion of EXPAR in the amplification system would reduce the overall robustness of the system. So we were forced to consider a different amplification system.

References

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1. Carter, J. G., Orueta Iturbe, L., Duprey, J. H. A., Carter, I. R., Southern, C. D., Rana, M., Whalley, C. M., Bosworth, A., Beggs, A. D., Hicks, M. R., & Tucker, J. H. R. (2021). Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR. Proceedings of the National Academy of Sciences, 118(35), e2100347118. https://doi.org/10.1073/pnas.2100347118

2. Funakoshi Co., Ltd. (n.d.). dsGreen: Kakusan geru senshoku shiyaku / riaru taimu PCR-yo SYBR®-kei keikō shikiso. https://www.funakoshi.co.jp/contents/67539

3. Özay, B., Murphy, S. D., Stopps, E. E., Gedeon, T., & McCalla, S. E. (2022). Positive feedback drives a secondary nonlinear product burst during a biphasic DNA amplification reaction. Analyst, 147(20), 4450-4461. https://doi.org/10.1039/D2AN01067D

4. FASMAC. (n.d.). DNA/RNA jutaku gosei seisei guredo no sentaku ni tsuite. https://fasmac.co.jp/dna_rna_purify_grade

TWJ-EXPAR

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Overview

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.

1. Preliminary Experiments

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.

2. Changing the Concentration of Template2

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.

3. Division

We conducted the experiments in two phases, as illustrated in the diagram below.

First Half

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.

Second Half

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.

Conclusion

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.

References

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1. 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

2. 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

ThisAmp

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Overview

ThisAmp is a modified version of the SDA reaction that incorporates the TWJ structure, and it was reported by Lee 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 ThisAmp, see Experiments_ThisAmp

The reaction mechanism of ThisAmp:

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).

1. Confirming the Need for Annealing

This experiment was conducted to determine whether the annealing process of target, template, and helper to form a three-strand complex, which is done in the prior study, is truly necessary. If the annealing step can be omitted, the reaction could begin with a simple mixture of these DNAs and enzymes, making the mechanism more accessible and convenient for use in household settings.

The fluorescence changes were plotted below.

In the paper with annealing operation, the amplification curves for target 1 fM and negative control (NC) were distinguishable. However, skipping the annealing step resulted in distinguishable amplification curves only up to target concentrations of 100 nM, which is approximately 10^8 orders of magnitude worse than the results presented in the original paper. Based on these results, it was concluded that the annealing process is necessary.

2. Preliminary Experiments

We attempted to verify the amplification mechanism described in the paper. While the paper used NEBuffer 3.1 as the buffer, we opted to use NEBuffer r3.1. The only difference between these buffers is that albumin in the buffers is recombinant in NEBuffer r3.1. Additionally, the paper employed vent (exo-) DNA polymerase, but we used Bst 2.0, which had already demonstrated amplification in EXPAR. To accommodate Bst 2.0, we replaced the ThermoPol Reaction Buffer with Isothermal Amplification Buffer.

The fluorescence changes obtained are shown in the graphs below. The left graph represents the results up to 60 min. While we conducted measurements at 60 min, an increase in fluorescence intensity was observed in less than 2 min. The right graph zooms in on the first 10 min of the reaction.

The amplification curves for 10 nM and NC can be distinguished; however, the difference in the starting time of amplification is less than 2 min. This means that with even a slight extension of the reaction time, it becomes difficult to differentiate between the two. This could lead to an increase in false positive rates when used in a home setting. Therefore, this issue needs to be addressed for more reliable detection.

3. Tuning of Template Concentration

To suppress the amplification of NC and increase the time gap between the start of amplification for the target and NC, we performed tuning of the template concentration.

The fluorescence changes were plotted below.

When the template concentration was lowered, it was observed that the amplification efficiency decreased, and at concentrations lower than 1/64 of the value used in the reference paper, no amplification could be detected within 60 min in SYBR Green Ⅰ fluorescence measurement. However, lowering the template concentration would not only slow the amplification speed but also reduce the amount of amplified product. Given the goal of amplifying and quantifying nucleic acids, lowering the template concentration was not desirable. Therefore, the template concentration was kept at 50 nM, as specified in the original paper.

4. Tuning of Polymerase Concentration

Tuning of polymerase concentration was performed to suppress NC amplification by decreasing the rate of amplification while maintaining the amount of amplified product, and to increase the difference between the time when target amplification begins and the time when NC amplification begins.

Fluorescence intensity was as shown in the left figure, and for polymerase concentration, the time until fluorescence intensity reached 8 * 10^5 is shown in the right figure. Error bars represent standard deviation.

The largest difference in the time required for the fluorescence intensity to reach $ 8 ¥times 10^5 $ between the target concentration of 100 pM and 0 M was observed when the polymerase concentration was 1/4 of the published data, 0.012 U/µL. The difference at this time was about 20 min. Based on this, we thought thatwe could solve the problem described in 2. by reducing the polymerase final concentration to 0.012 U/µL.

5. Re-experiment under Tuned Conditions

To confirm the LoD of this system, the experiment was repeated with the conditions obtained in section 4. and 5.

The fluorescence changes were plotted below.

The LoD was 10 nM in 2. before tuning, but after tuning, the LoD was 100 pM.

Conclusion

ThisAmp had a LoD of 100 pM after optimization. This reaction is characterized by the fact that it proceeds at a constant 55 ℃ and requires no temperature change. ThisAmp is useful in that it can form a TWJ and amplify miRNAs, but it is not suitable for miRNA amplification due to its long target length (59 mer) . In addition, since the target, template, and helper must be annealed before adding the enzyme, it is difficult to connect other amplification mechanisms before this reaction considering 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

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1. Lee, S., Jang, H., Kim, H. Y., & Park, H. G. (2020). Three-way junction-induced isothermal amplification for nucleic acid detection. Biosensors and Bioelectronics, 147, 111762. https://doi.org/10.1016/j.bios.2019.111762

TWJ-Toehold

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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).

1. Preliminary Experiments

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.

The fluorescence changes were plotted below.

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

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

The fluorescence changes were plotted below.

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

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

Fluorescence changes were obtained as shown in the graph below.

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

TWJ-2cycle

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Overview

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.

1. Target miRNA, helper DNA, and template form a TWJ complex.
2. Polymerase elongates the 3' side of helper.
3. Nickase introduces a nick.
4. Polymerase elongates from the nick while stripping off the previous strand.
5. 3. - 4. are repeated, and a large number of strands on the 3' side of the helper (trigger) are generated.
6. Trigger forms a complex with hairpin probe and MB probe.
7. Nickase cleaves the MB probe, and the Fluorescent and Quencher probes are separated. The separated Fluorescent probe becomes fluorescent.
8. Repeating steps of 6. - 7, MB probes are cleaved one after another, and the fluorescence intensity increases.

The series of reactions proceed at an isothermal temperature of 37 ℃

1. Preliminary Experiments

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.

2. Changing Buffer

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.

3. Tuning of the Concentration of Polymerase and Nickase

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.

Table 1: Maximum S/N ratio recorded during the reaction time

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.

4. Specificity

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 reference [1], 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, see engineering, Model_Specificity.

Conclusion

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.

References

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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

2. New England Biolabs. (n.d.). Bst DNA polymerase, large fragment. https://www.neb.com/ja-jp/products/m0275-bst-dna-polymerase-large-fragment

3. New England Biolabs. (n.d.). Nt.BbvCI (nicking endonuclease). https://www.neb.com/ja-jp/products/r0632-ntbbvci

Designed sequences

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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 ^DS1.

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, see Proposed Implementation_biomarker

Designing helper for biomarkers

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.

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

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.

For each biomarker, the following measurements were obtained.

biomarker1

Biomarker2

Biomarker3

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.

Designing helper for biomarkers

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.

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

Biomarker2

Biomarker3

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.

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, see Model_Sequencedesign

References

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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

Multistep-SDA

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Overview

Strand Displacement Amplification (SDA) is a method to produce a large amount of ssDNA by using DNA polymerase with strand displacement activity and nickase, which recognizes dsDNA and puts a nick on one of the strands. We focused on the system ¹ developed by Dr. Komiya at JAMSTEC. This system is a multi-step combination of SDA reactions to amplify ssDNA. The SDA reaction is originally subject to amplification even in negative control, but in this system, negative control is suppressed by various innovations. We call this system “multistep-SDA” and conducted experiments.
For more information about the principle of multistep-SDA, see Proposed Implementation_Amplification
For more information about the actual experimental procedure of multistep-SDA, see Experiments_Multistep-SDA

1. Preliminary Experiments

We attempted to confirm the amplification from single stranded nucleic acid by the mechanism described in the paper.
The paper uses NEBuffer 2 as the buffer, but we used NEBuffer 2.1. The only difference between NEBuffer 2 and NEBuffer 2.1 is whether DTT or albumin is used in the buffer, which does not seem to affect the enzyme activity ². In addition, in the paper, the 3' ends of the template DNA strands are chemically modified with carboxytetramethylrhodamine (TAMRA). We used ssDNA without this modification as the template. Fluorescence intensity was measured using MB as in the paper.

The fluorescence changes were plotted below.

It could be distinguished from NC if the target concentration was at least 100 pM. In the paper, no amplification of NC was observed after 150 min, but in this experiment, amplification started at about 25 min.
We conducted further experiments to elucidate the cause of the apparently faster amplification than in the paper.

2. Pursuing the Cause of Amplification in NC

We wanted to clarify the cause of the apparent faster amplification than in the paper. To determine which step was responsible, we performed experiments with each step of multistep-SDA, as well as with two or three steps of multistep-SDA connected together. Since the MB used in the paper cannot detect the amplification products of the first and second steps of SDA, we also measured the fluorescence intensity using SYBR Green I or SYBR Green II. Additionally, we performed experiments using the optimized buffer in the paper (we call it LT Buffer) instead of NEBuffer 2.1.

The fluorescence changes were plotted below.

When only step1 and step2 were used, the amplification speed was slow and amplification of NC was not observed, but amplification of target was also almost unobservable. It was confirmed that amplification efficiency increased with the number of steps, but when two or more steps were linked, amplification of NC was observed. Also, when NEBuffer 2.1 and LT Buffer were compared in step123, no significant difference was observed.

Consult with Extra Adviser Dr. Komiya

In the paper, no amplification of NCs was observed in the 3step-SDA reaction, but in our experiment, amplification of NCs was observed by connecting multiple steps of SDA. We asked Dr. Komiya, the author of the paper and our extra adviser, for tips. He told us that it is important that the 3' ends of template DNA are chemically modified with carboxytetramethylrhodamine (TAMRA) .

3. Follow-up Experiment with TAMRA Modified Template DNA Strands

Based on the advice, we performed follow-up experiments with template DNA strands whose 3' ends are chemically modified with TAMRA. Fluorescence intensity was measured using MB like in the paper.

Within 200 min of observation, amplification was observed at target concentrations of 10 nM or higher for only step3 and step23, and at target concentrations of 100 pM or higher for step123. In all cases, NC amplification was suppressed.

By using a template with TAMRA modification at the 3' end, amplification was confirmed within 200 min of the observation when the target concentration was 10 nM or higher for only step3 and step23. For step 123, amplification was confirmed when the target concentration was 100 pM or higher. In all cases, amplification of NCs was not confirmed during the observation time.

Conclusion

By using a template with TAMRA modification at the 3' end to link multiple stages of SDA, it was confirmed that target amplification could be efficiently achieved while NC amplification was suppressed. The increase in amplification efficiency by increasing the number of stages was also confirmed. However, amplification was observed within 200 min only when the target concentration was 100 pM or higher, and this mechanism cannot be used in POIROT, which aims for detection in the fM order. Therefore, Multistep-SDA must be combined with other amplification mechanisms.

Multistep-SDA is a system that uses short nucleic acids as input, and in principle, any sequence of nucleic acids can be amplified by changing the template sequence. In addition, this system is characterized by the fact that it proceeds at 37 ºC without the need for temperature changes. Furthermore, it is also possible to amplify dsDNA by changing the template sequence and eliminating the nicking site. Therefore, multistep-SDA is a useful system that can be easily connected to other amplification methods that proceed at 37 °C and to the CRISPR-Cas system that targets dsDNA.

References

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1. Komiya, K., Noda, C. & Yamamura, M. (2024). Characterization of Cascaded DNA Generation Reaction for Amplifying DNA Signal.New Gener. Comput. 42, 237-252. https://doi.org/10.1007/s00354-024-00249-2

2. New England Biolabs. (2013). Why did you remove DTT from your restriction enzyme buffers? https://www.neb.com/ja-jp/faqs/2013/02/28/why-did-you-remove-dtt-from-your-restriction-enzyme-buffers

Abstract

Abstract

Abstract