iGEM 2020 — Team Leiden

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# Team:Leiden/Notebook

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iGem Leiden 2020

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## Rapidemic

[Skip to main content](#slide1-chapter1)

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PROJECT

[Description](https://2020.igem.org/Team%3ALeiden/Description)
[Results](https://2020.igem.org/Team%3ALeiden/Results)
[Proof of Concept](https://2020.igem.org/Team%3ALeiden/Proof_Of_Concept)
[Engineering](https://2020.igem.org/Team%3ALeiden/Engineering)
[Models](https://2020.igem.org/Team%3ALeiden/Model)
[Hardware](https://2020.igem.org/Team%3ALeiden/Hardware)
[Contribution](https://2020.igem.org/Team%3ALeiden/Contribution)

LAB

[Experiments](https://2020.igem.org/Team%3ALeiden/Experiments)
[Parts](https://2020.igem.org/Team%3ALeiden/Parts)
[Notebook](https://2020.igem.org/Team%3ALeiden/Notebook)
[Safety](https://2020.igem.org/Team%3ALeiden/Safety)

HUMAN PRACTICES

[Integrated Human Practices](https://2020.igem.org/Team%3ALeiden/Human_Practices)
[Education & Public Engagement](https://2020.igem.org/Team%3ALeiden/Education)
[Implementation](https://2020.igem.org/Team%3ALeiden/Implementation)
[Entrepreneurship](https://2020.igem.org/Team%3ALeiden/Entrepreneurship)
[Inclusion](https://2020.igem.org/Team%3ALeiden/Inclusion)

OUTREACH

[Featured](https://2020.igem.org/Team%3ALeiden/Featured)
[Newsletter](https://2020.igem.org/Team%3ALeiden/Newsletter)
[Crowdfunding](https://2020.igem.org/Team%3ALeiden/Crowdfunding)

TEAM

[Team](https://2020.igem.org/Team%3ALeiden/Team)
[Collaborations](https://2020.igem.org/Team%3ALeiden/Collaborations)[Attributions](https://2020.igem.org/Team%3ALeiden/Attributions)[Sponsors](https://2020.igem.org/Team%3ALeiden/Sponsors)

AWARDS

[Prizes](https://2020.igem.org/Team%3ALeiden/Prizes)
[Medal Criteria](https://2020.igem.org/Team%3ALeiden/Medals_And_Awards)

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* AWARDS
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  + [Medal Criteria](https://2020.igem.org/Team%3ALeiden/Medals_And_Awards)

# Notebook

Below you can find the procedures of the performed experiments in chronological order, categorized into three main categories: (1) Recombinase polymerase amplification (RPA), (2) Guanine-quadruplex (GQ)-catalyzed oxidation and (3) Coupling RPA, LSDA and oxidation. The experimental protocols can be found on the Experiments page, while the adjustments to the protocols are addressed on this page in the lab notes of the experiment. The lab notebook was written using Benchling.

RPA

GQ-catalysed Oxidation

Coupling the reactions

Other Experiments

RPA

If the pdf doesn't show, click [here](https://static.igem.org/mediawiki/2020/d/d7/T--Leiden--RPA.pdf) to download the pdf!

[Click here](https://static.igem.org/mediawiki/2020/a/a3/T--Leiden--1RPA.zip) to download the raw data mentioned in the notebook.

GQ-Oxidation

If the pdf doesn't show, click [here](https://static.igem.org/mediawiki/2020/1/1f/T--Leiden--GQ-catalyzed_oxidation.pdf) to download the pdf!

[Click here](https://static.igem.org/mediawiki/2020/4/4d/T--Leiden--2_GQ.zip) to download the raw data mentioned in the notebook.

Coupling the reactions

If the pdf doesn't show, click [here](https://static.igem.org/mediawiki/2020/3/36/T--Leiden--Coupling_the_reactions.pdf) to download the pdf!

[Click here](https://static.igem.org/mediawiki/2020/5/5b/T--Leiden--3_Coupling_the_reactions.zip) to download the raw data mentioned in the notebook.

Other experiments

If the pdf doesn't show, click [here](https://static.igem.org/mediawiki/2020/e/e8/T--Leiden--InfA-H1N1_LAMP_Trial_2020-08-20_%28etr_Z92wKivM%29.pdf) to download the pdf!

### About Us

We are the 2020 team of iGEM Leiden. With an interdisciplinary team of students we aim to develop a point-of-care rapid diagnostic tool for infectious diseases!

### Follow Us

### Contact Us

iGEM Team Leiden
Sylviusweg 72, 2333BE Leiden
igem@science.leidenuniv.nl

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---

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# Team:Leiden/Engineering

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iGem Leiden 2020

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## Rapidemic

[Skip to main content](#slide1-chapter1)

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[HOME](https://2020.igem.org/Team%3ALeiden)

PROJECT

[Description](https://2020.igem.org/Team%3ALeiden/Description)
[Results](https://2020.igem.org/Team%3ALeiden/Results)
[Proof of Concept](https://2020.igem.org/Team%3ALeiden/Proof_Of_Concept)
[Engineering](https://2020.igem.org/Team%3ALeiden/Engineering)
[Models](https://2020.igem.org/Team%3ALeiden/Model)
[Hardware](https://2020.igem.org/Team%3ALeiden/Hardware)
[Contribution](https://2020.igem.org/Team%3ALeiden/Contribution)

LAB

[Experiments](https://2020.igem.org/Team%3ALeiden/Experiments)
[Parts](https://2020.igem.org/Team%3ALeiden/Parts)
[Notebook](https://2020.igem.org/Team%3ALeiden/Notebook)
[Safety](https://2020.igem.org/Team%3ALeiden/Safety)

HUMAN PRACTICES

[Integrated Human Practices](https://2020.igem.org/Team%3ALeiden/Human_Practices)
[Education & Public Engagement](https://2020.igem.org/Team%3ALeiden/Education)
[Implementation](https://2020.igem.org/Team%3ALeiden/Implementation)
[Entrepreneurship](https://2020.igem.org/Team%3ALeiden/Entrepreneurship)
[Inclusion](https://2020.igem.org/Team%3ALeiden/Inclusion)

OUTREACH

[Featured](https://2020.igem.org/Team%3ALeiden/Featured)
[Newsletter](https://2020.igem.org/Team%3ALeiden/Newsletter)
[Crowdfunding](https://2020.igem.org/Team%3ALeiden/Crowdfunding)

TEAM

[Team](https://2020.igem.org/Team%3ALeiden/Team)
[Collaborations](https://2020.igem.org/Team%3ALeiden/Collaborations)[Attributions](https://2020.igem.org/Team%3ALeiden/Attributions)[Sponsors](https://2020.igem.org/Team%3ALeiden/Sponsors)

AWARDS

[Prizes](https://2020.igem.org/Team%3ALeiden/Prizes)
[Medal Criteria](https://2020.igem.org/Team%3ALeiden/Medals_And_Awards)

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[HOME](https://2020.igem.org/Team%3ALeiden)

* PROJECT
  + [Description](https://2020.igem.org/Team%3ALeiden/Description)
  + [Results](https://2020.igem.org/Team%3ALeiden/Results)
  + [Proof of Concept](https://2020.igem.org/Team%3ALeiden/Proof_Of_Concept)
  + [Engineering](https://2020.igem.org/Team%3ALeiden/Engineering)
  + [Models](https://2020.igem.org/Team%3ALeiden/Model)
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* HUMAN PRACTICES
  + [Integrated Human Practices](https://2020.igem.org/Team%3ALeiden/Human_Practices)
  + [Education & Public Engagement](https://2020.igem.org/Team%3ALeiden/Education)
  + [Implementation](https://2020.igem.org/Team%3ALeiden/Implementation)
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* OUTREACH
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* AWARDS
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# Engineering

Welcome to the engineering page! Here, we describe how we developed and tested the bio-molecular reactions of our technology. During this process of engineering, successful results were obtained, but also new challenges were discovered. Nevertheless, the drawbacks forced us to come up with new ideas and initiated new cycles of engineering, ultimately driving our project forward. While the exact experiments and results are described on the [Results](https://2020.igem.org/Team%3ALeiden/Results) page, on this page we outline the underlying thought process that led to our final engineering success.

Summary

Design of the mechanism

Establish functioning reaction conditions

Reduce false-positive signals

Future Prospect

References

![Background](https://static.igem.org/mediawiki/2020/3/3c/T--Leiden--EngineeringBackground-min.jpg)

![Gear 1](https://static.igem.org/mediawiki/2020/5/5d/T--Leiden--gear1.png)

![Gear 2](https://static.igem.org/mediawiki/2020/8/8d/T--Leiden--gear2.png )

![Gear 4](https://static.igem.org/mediawiki/2020/1/1f/T--Leiden--gear4.png)

![Gear 3](https://static.igem.org/mediawiki/2020/4/43/T--Leiden--gear3.png)

## Summary

Driven by the urgent need for innovative tests to effectively contain future outbreaks of infectious diseases, we designed a label-free visual detection method as a simple and low-cost alternative to the existing detection techniques. For the proof-of-concept of the new technology, we used the genomic DNA of *Saccharomyces cerevisiae* (*S. cerevisiae*) as a model organism, as the use of the complete genome of a pathogenic organism was not considered safe. To deliver the proof of concept, we went through three cycles of engineering (research - brainstorm - design - test - learn - improve...). We started the process by researching the shortcomings of existing detection methods and brainstorming which conditions should be met by our detection method. The main criteria were versatility, low-cost, easy fabrication and point-of-care use (see Fig. 1). With these criteria in mind, a new bio-molecular detection scheme was designed and tested. We found out, however, that the technology could not successfully detect the genetic material with the initial protocols that were used. Therefore, we went through a second engineering cycle, during which we developed two strategies to eliminate the factor that initially inhibited the detection. At last, the combination of these two strategies successfully enabled the detection of *S. cerevisiae* DNA.

The third and last cycle of our engineering process was focused on a challenge that remains an active point-of-interest. In this cycle, we were confronted with the occurrence of false-positive signals whose origin was traced back to the first of the three main reactions in our detection method. For the full development of the system, further innovation of this first step will be crucial to reduce the false-positive rate and to test the specificity, sensitivity, and the limit of the technology. Nevertheless, here we hope to inspire you with the insights that we gained throughout our engineering journey: click on the gears above to read more about each step on the way to a working detection mechanism.

#### Engineering achievements in short

+ We designed a label-free visual detection mechanism (Cycle 1)
+ We experimentally engineered functioning reaction conditions for this method (Cycle 2)
+ We characterized a [New Part](https://2020.igem.org/Team%3ALeiden/Parts)
+ We improved an [Existing Part](https://2020.igem.org/Team%3ALeiden/Parts)

![](https://static.igem.org/mediawiki/2020/0/0c/T--Leiden--Gear1_with_text.png)

## Cycle 1: The design of the mechanism

### Research existing detection techniques

*Text adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08).*

With the aim to develop a rapid, accurate, and accessible diagnostic kit, [we narrowed our focus to point-of-care nucleic acid-based tests.](https://2020.igem.org/Team%3ALeiden/Description) Below, we summarize techniques that form the basis of currently existing kits and highlight several areas of improvement.

Several nucleic acid amplification techniques have been developed as point-of-care alternatives to the laboratory-restricted PCR tests1. Many of those techniques use isothermal amplification methods. These are enzyme-based methods that work at a low and constant temperature. Examples of isothermal amplification methods are loop-mediated amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), strand-displacement amplification (SDA), isothermal exponential amplification reaction (EXPAR), and recombinase polymerase amplification (RPA)2-7. Among other methods, RPA was found to be a promising method for versatile point-of-care applications due to its low reaction temperature and relatively simple primer design7,8. In the RPA reaction, a recombinase is used to assist the binding of two primers to the template DNA upon which a strand-displacement polymerase amplifies the target sequence.

DNA and RNA targets of various organisms have been successfully amplified with RPA for rapid point-of-care diagnostics applications9. End-point detection is usually performed using lateral flow strips or fluorescent probes9. However, these existing methods may impair the simplicity of the test, as detection by lateral flow requires oligonucleotide labeling and immobilization of antibodies, whereas fluorescent detection may require a specialized instrument to read the output signal10. In addition, the colloidal gold and antibodies often used in lateral flow strips increase the cost of the test substantially11. Therefore, new, simple, and low-cost detection methods that can be coupled to isothermal amplification reactions would be valuable alternatives to the current techniques.

There has been an increasing interest in the use of DNA enzymes (DNAzymes) with peroxidase-mimicking activity for molecular diagnostics12,13. DNAzymes are short strands of DNA with an enzyme-like activity. For instance, DNAzymes  with a peroxidase-mimicking activity can be employed to catalyze an oxidation-based color reaction. Their high chemical and thermal stability compared to protein enzymes make DNAzymes suitable for point-of-care testing. In addition, DNAzymes are amenable to various amplification methods, which can be employed to improve the sensitivity of the assays12. DNAzyme-mediated oxidation coupled to isothermal amplification methods has been described in previous studies14,15. Li et al. (2019) integrated a G3 DNAzyme with EXPAR for sensitive nucleic acid detection14. However, their mechanism of amplification severely limits the choice for a target sequence. Alternatively, Wang et al. (2019) exploited asymmetric amplification to couple RPA with DNAzyme-catalyzed detection15. The asymmetric RPA resulted in the amplification of DNAzyme sequences that contained a large strand of bases adjacent to their 5’ end, which may possibly hamper with the peroxidase-mimicking activity of the DNAzyme. Moreover, asymmetric amplification methods such as asymmetric RPA often suffer from low amplification efficiency, case-specific optimization requirement, and generation of non-specific product(s)16,17.

### Imagine what it takes to make a good point-of-care detection method

With our literature research as a basis, we brainstormed what properties belong to a versatile low-cost point-of-care detection system. The mind map below illustrates what we believe is essential for the design of a simple point-of-care diagnostic test, as well as potential practical solutions, to realize these aspects (Fig. 1). During the literature search, we had gained an interest in DNAzymes as reporters due to their nature as DNAs, which allows them to be produced in amplification reactions. This led us to the following research question: *Can we design a detection system based on nucleic acid amplification that produces single-stranded DNAzyme sequences to catalyze a color reaction?*

![](https://static.igem.org/mediawiki/2020/f/f3/T--Leiden--simplicity-min.png)

**Fig. 1 Brainstorm (mind map) for the development of a new simple point-of-care detection system.** We imagined a simple point-of-care detection system that is cheap, easy-to-use, and easy to fabricate. The method should be based on nucleic acid amplification so that it is easily adaptable to new targets by simply changing its primers. Amplification at a low and constant temperature, as with recombinase polymerase amplification (RPA), eliminates the need for specialized heating equipment. Additionally, we highlight the low-cost and simplicity aspects with the use of a nucleic acid-based reporter such as a DNAzyme, instead of fluorescent labels, antibodies, and colloidal gold, which are commonly used in several existing tests (lateral flow). Lastly, the test results should be presented in such a way that no specialized readout instrument is needed, for instance with a colorimetric readout.

### Design the detection mechanism

After reflecting on the necessary properties for a point-of-care diagnostic kit, we designed Rapidemic, a label-free colorimetry-based detection method. This assay combines two amplification reactions with a DNAzyme-based color reaction. First, a target sequence is exponentially amplified using recombinase polymerase amplification (RPA) (Fig. 2). Subsequently, single-stranded DNA sequences of the DNAzyme (GQ) are produced with linear strand-displacement amplification (LSDA). Lastly, the DNAzymes catalyze an oxidation reaction that yields a color change. In this way, the specific amplification of a target sequence is coupled to an easy readout that confirms the presence of genetic material.

![](https://static.igem.org/mediawiki/2020/1/1e/T--Leiden--Results1_Hemin-GQ-min.png)

**Fig. 2 Amplification scheme of the technology.** Selective exponential amplification of a target sequence, followed by strand-displacement amplification leads to the production of single-stranded GQ DNAzyme DNAs.

Detailed description: how our technology detects target sequence
     +

---

### (1) Recombinase polymerase amplification (RPA)

Because the amount of genetic material of a pathogen in a human sample can be very low, it has to be amplified in order to be detectable. Therefore, targeted amplification occurs in the first step of the detection method using the recombinase polymerase amplification (RPA) technique, a method performed at a constant and low temperature. This provides our technique with a benefit as it does not require specialized or energy-consuming equipment for cycling different temperatures, as is the case for PCR-based methods.

We designed specific primers to target genomic DNA of *Saccharomyces cerevisiae*. The reverse primer contained an overhang at its 5' end, which consisted of the reverse complementary sequence of a nickase recognition site (N') and a GQ sequence (G') (Fig. 3). This overhang is important to couple RPA to the second and third step in the detection method.

![](https://static.igem.org/mediawiki/2020/c/cc/T--Leiden--Experiments_figure1_RPA.png)

**Fig. 3 RPA with a non-tailed forward primer and a tailed reverse primer.** The tail of the reverse primer contains the reverse complementary sequences of a nickase recognition site (N') and a GQ sequence (G'). As a result, the top strand of the amplification product contains a nickase recognition site (N) and a GQ sequence (GQ).

### (2) Linear strand-displacement amplification (LSDA)

The second reaction in the detection method, also known as linear strand-displacement amplification (LSDA), couples the amplification and the color reaction. In short, a nicking endonuclease (nickase) recognizes a specific site in the amplified RPA product and makes a single-stranded cut in the top DNA strand (Fig. 4). Subsequently, a strand-displacement DNA polymerase binds at that site and elongates the top strand starting from the 3' end at the nickase cut site. In doing so, the GQ sequence is displaced from the double-stranded structure.

![](https://static.igem.org/mediawiki/2020/f/f7/T--Leiden--LSDA-animation.gif)

**Fig. 4 Linear strand-displacement amplification (LSDA) mechanism.** The nickase binds at its recognition site in the RPA product and makes a single-stranded cut in the top strand of the double-stranded DNA complex. Then, a strand-displacement DNA polymerase binds at the cut site and elongates the top strand starting from the 3' end.

### (3) Guanine-quadruplex (GQ)-catalyzed oxidation

The third and last step in the detection method is the GQ-catalyzed oxidation reaction. The single-stranded GQ DNA sequence that was amplified in the previous reaction forms a three-dimensional structure by binding to potassium ions and hemin (Fig. 5a). This GQ structure possesses an enzyme-like activity and is thus referred to as a DNAzyme. The DNAzyme catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine sulfate (TMB) in the presence of hydrogen peroxide (H2O2), thereby producing a color change (Fig. 5b). Based on this color change, the user of the diagnostic test can confirm whether the human sample contained the genetic material of the pathogen-of-interest; if the color changes from transparent to blue, the patient is infected.

![](https://static.igem.org/mediawiki/2020/6/67/T--Leiden--Engineering8_Experiments_GQ-Oxidation.jpg)

**Fig. 5 Guanine-quadruplex (GQ)-catalyzed TMB oxidation. a)** Single-stranded GQ sequences form a GQ structure with peroxidase-mimicking activity upon binding potassium ions and hemin. **b)** The GQ DNAzyme catalyses the oxidation of TMB in the presence of hydrogen peroxide (H2O2). First, an intermediate product, a radical cation-TMB/diimine complex, is formed with a blue color. The addition of sulfuric acid then pushes the TMB oxidation reaction to the second reaction product, a diimine with yellow color.

### Test and learn: an inhibitor prevented the change of color

Once we had designed the detection scheme, we built the protocols for the three bio-molecular reactions involved based on methods reported in literature18-21. The reactions were coupled by using the product of each preceding reaction as a template or catalyst in the next reaction. Although each reaction was successful on its own, a color change could not be obtained upon coupling the three reactions. This forced us to reflect on possible causes of the absent color change.

It was discovered that a rather high concentration of the reducing agent dithiothreitol (DTT) was present in the RPA reaction mix. This chemical was passed along with the RPA product to the last reaction where it inhibited the oxidation reaction that should yield a color change. We learned that dilution and/or inactivation of DTT was essential for the detection method to work. We tried to tackle this challenge in the next engineering cycle.

![](https://static.igem.org/mediawiki/2020/4/4f/T--Leiden--Gear2_with_text.png)

## Cycle 2: Establish functioning reaction conditions

### Research how DTT inhibited the color change

DTT, the compound that inhibited the GQ-catalyzed oxidation reaction, is a reducing agent that is often added to enzyme mixes to improve protein stability. It prevents the formation of intramolecular and intermolecular disulfide bonds between cysteine residues of proteins by keeping the proteins’ thiol groups in their reduced state (Fig. 6)22. With its reducing power, DTT had disabled the formation of a color change in the last step of the detection method. However, DTT's reducing power is known to be weak in acidic conditions because DTT's reactive negatively charged thiolate is absent at low pH23. This knowledge helped us in designing strategies to eliminate DTT's inhibiting effect.

![](https://static.igem.org/mediawiki/2020/b/b6/T--Leiden--DTT_Figure5.png)

**Fig. 6 DTT reduces protein thiol groups.** Figure adapted from [https://commons.wikimedia.org](https://commons.wikimedia.org/wiki/File%3ADisulfide_reduction_by_DTT-2.png).

### Imagine and design strategies to eliminate inhibition by DTT

![](https://static.igem.org/mediawiki/2020/b/b5/T--Leiden--dilution_figure.png)

**Fig. 7 Optimization of the dilution rates.**

We thought of two strategies that might resolve the inhibition of the color change: (1) decrease the concentration of DTT, and (2) change the acidity of the oxidation buffer to decrease DTT's reducing power. Firstly, we designed new oxidation buffers that inactivate DTT but still accommodate the DNAzyme's activity. These included a phosphate buffer and a phosphate citrate buffer with varying pH (pH 3-6). Secondly, we designed dilution schemes that decrease the concentration of DTT in the oxidation reaction. Since the detection method consists of three reactions, there are two dilution steps (Fig. 7). The dilution rates from RPA to LSDA and from LSDA to oxidation were optimized, so that (1) the DTT from the RPA mix is diluted 800-fold when it reaches the oxidation reaction chamber, while (2) enough product of each preceding reaction is passed on to the next for the reactions to be operable.

![](https://static.igem.org/mediawiki/2020/b/b5/T--Leiden--dilution_figure.png)

**Fig. 7 Optimization of the dilution rates.**

### Test and learn: successful reaction conditions established! However...

In a second attempt to combine the three reactions, we tested whether the new acidic buffer solutions and the dilution schemes enabled the oxidation reaction. A phosphate-citrate buffer with pH 3.8 was found to be effective in inactivating DTT. Only with the combination of the two strategies, a strong color change could be observed (Fig. 8). This suggested that the three reactions of the detection method were coupled successfully!

A second observation during this experiment was the occurrence of false-positive signals in the absence of target DNA, which originated from primer-associated (off-target) amplification in the first step of the detection method. The reduction of false-positive signals became the next priority in the engineering process of this technology, and is the subject of the next cycle.

![](https://static.igem.org/mediawiki/2020/a/aa/T--Leiden--Reaction_Figure7.png)

**Fig. 8 Coupling of RPA, LSDA, and oxidation with the updated dilution scheme and buffer produced an observable color change.** "-" indicates negative control (no LSDA product), "+" indicates the reaction with LSDA product.

#### [Contribution to the Registry of Standard Biological Parts](https://2020.igem.org/Team%3ALeiden/Parts)

![](https://static.igem.org/mediawiki/2020/1/1f/T--Leiden--Comparing_DNAzymes_over_time_650nm-min.png)

**Fig. 9 Peroxidase activity of three DNAzymes: BBa\_K1614007 (original part), BBa\_K3343001 (improved part with additional adenine) and BBa\_K3343000 (DNAzyme with highest activity reported in literature).**

For the color reaction in the detection method, we searched for suitable peroxidase-mimicking DNAzyme sequences in the Registry of Standard Biological Parts. We found one DNAzyme ([BBa\_K1614007](http://parts.igem.org/Part%3ABBa_K1614007)), filed by iGEM team Heidelberg 2015, that could be applied for our technique. However, this DNAzyme did not show any peroxidase activity in our experimental conditions. Presumably, this was the result of the pH at which the oxidation reaction was performed; the DNAzyme activity is very low at pH 621. The optimal pH of the part is approximately 8.5, and its activity sharply decreases within the pH range of 8 to 621,24. The detection mechanism requires a DNAzyme that can work at pH 3.8, as the oxidation buffer with this pH effectively weakened the inhibiting effect of DTT. Inspired by literature, we added an extra adenine to the 3' end of the DNAzyme sequence and successfully broadened its pH range (Fig. 9)21.

![](https://static.igem.org/mediawiki/2020/1/1f/T--Leiden--Comparing_DNAzymes_over_time_650nm-min.png)

**Fig. 9 Peroxidase activity of three DNAzymes: BBa\_K1614007 (original part), BBa\_K3343001 (improved part with additional adenine) and BBa\_K3343000 (DNAzyme with highest activity reported in literature).**

![](https://static.igem.org/mediawiki/2020/4/49/T--Leiden--Gear3_with_text.png)

## Cycle 3: Reduce false-positive signals

### Research the causes of false-positive amplification signals & imagine how to counteract these mechanisms

False-positive signals in amplification reactions may be the result of non-specific amplification, for instance, caused by the annealing of the primers at different sites on the target DNA or the primer-associated amplification that does not involve the target DNA. In our case, the observed false-positive signals originated from the formation of secondary structures by the primers of primer-primer interactions. A common approach to increase the specificity of amplification reactions is the addition of small chemicals, such as dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG)25. As PEG was already present in our RPA reaction mix, we wondered whether the addition of DMSO could reduce the false-positive rate. DMSO enhances the specificity of amplification reactions by disrupting the formation of weaker secondary structures of DNA while allowing the stronger specific primer binding to occur26,27. Therefore, the addition of DMSO to the RPA reaction could prevent (self-)annealing of primers or primer-primer interactions.

### Design and test strategies to reduce off-target amplification

Since a balance should be obtained between reducing the off-target amplification and maintaining the targeted amplification, different concentrations of DMSO were included in the RPA reaction mix. The RPA reactions were monitored in a real-time manner to test the effect of the different concentrations of DMSO on off-target amplification.

### Understand the effect of DMSO, integrate into the model simulation and predict

After obtaining measurement results from different DMSO concentrations, we were faced with the challenge to select an optimal concentration to include in our tests. Our *in silico* analysis identified a DMSO concentration of 1.33% to be optimal for maximizing the difference in color between the positive and the negative tests, i.e. maximizing the difference in endpoint signal intensity (absorbance at 650 nm). This concentration of DMSO is marked by the dashed line in Fig. 10. Nevertheless, this DMSO concentration did not minimize the endpoint false-positive signal intensity. Further analysis revealed 2.5% DMSO (pointed by the red triangle) to be the lowest DMSO concentration that can minimize endpoint false-positive signals to a level comparable to the blank control, despite not being the concentration that can maximize the difference between true-positive and false-positive signals.

![](https://static.igem.org/mediawiki/2020/c/ca/T--Leiden--False_Positives.png)

**Fig. 10 Our model identified 2.5% DMSO concentration as the optimal DMSO concentration that minimizes the endpoint false-positive signals (marked by the red arrow).** This concentration can thus be used to minimize the false-positive signal as much as possible.

In the lab, we evaluated the effectivity of 2.5% DMSO in RPA as a strategy to minimize false-positive signals. Preliminary analysis using RT-RPA showed how the inclusion of 2.5% DMSO in the RPA mix could effectively reduce background (no-template) amplification without severely impacting target-specific amplification. However, further evaluation of the resulting detection scheme (RPA with 2.5% DMSO followed by LSDA and GQ oxidation) showed how the new reaction settings could barely distinguish true-positive signals from the background colorimetry signals. We thus learned that this strategy, though effective in attenuating the severity of false-positive signals, was not sufficient to allow for distinction between the true-positive signals and background including residual false-positive signals.

![](https://static.igem.org/mediawiki/2020/9/9d/T--Leiden--Gear4_with_text.png)

## Future Prospect

The 'open ending' of the last engineering cycle has led us to a new engineering challenge; further innovation of the first step in our detection system (RPA) will be essential to reduce the false-positive rate of the technology. We may focus on alternative enhancing chemicals that do not interfere with the oxidation reaction, as well as improve the primer sequences to make them less prone to off-target interactions. Ultimately, we plan to develop the technology further to enhance its specificity, sensitivity, and limit of detection. So, stay tuned for our future journey through the cycles of engineering!

## References

1. Yan, L. et al. Isothermal amplified detection of DNA and RNA. Molecular BioSystems 10, 970–1003 (2014).
2. Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000).
3. Guatelli, J. C. et al. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. U. S. A. 87, 1874–8 (1990).
4. Ali, M. M. et al. Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chem. Soc. Rev. 43, 3324–41 (2014).
5. Walker, G. T. et al. Strand displacement amplification - an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20, 1691–1696 (1992).
6. Van Ness, J., Van Ness, L. K. & Galas, D. J. Isothermal reactions for the amplification of oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 100, 4504–4509 (2003).
7. Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, e204 (2006).
8. Zanoli, L. M. & Spoto, G. Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors 3, 18–43 (2013).
9. Li, J., Macdonald, J. & Von Stetten, F. Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst 144, 31–67 (2019).
10. Kozwich, D. et al. Development of a novel, rapid integrated Cryptosporidium parvum detection assay. Appl. Environ. Microbiol. 66, 2711–2717 (2000).
11. Yetisen, A. K., Akram, M. S. & Lowe, C. R. Paper-based microfluidic point-of-care diagnostic devices. Lab on a Chip 13, 2210–2251 (2013).
12. Peng, H. et al. DNAzyme-Mediated Assays for Amplified Detection of Nucleic Acids and Proteins. Analytical Chemistry 90, 190–207 (2018).
13. Roembke, B. T., Nakayama, S. & Sintim, H. O. Nucleic acid detection using G-quadruplex amplification methodologies. Methods 64, 185–198 (2013).
14. Li, R., Liu, Q., Jin, Y. & Li, B. G-triplex/hemin DNAzyme: An ideal signal generator for isothermal exponential amplification reaction-based biosensing platform. Anal. Chim. Acta 1079, 139–145 (2019).
15. Wang, Y., Li, X., Xi, D. & Wang, X. Visual detection of: Fusarium proliferatum based on asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme. RSC Adv. 9, 37144–37147 (2019).
16. Heiat, M., Ranjbar, R., Latifi, A. M., Rasaee, M. J. & Farnoosh, G. Essential strategies to optimize asymmetric PCR conditions as a reliable method to generate large amount of ssDNA aptamers. Biotechnol. Appl. Biochem. 64, 541–548 (2017).
17. Sanchez, J. A., Pierce, K. E., Rice, J. E. & Wangh, L. J. Linear-After-The-Exponential (LATE)-PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proc. Natl. Acad. Sci. U. S. A. 101, 1933–1938 (2004).
18. Nie, J. et al. A G-quadruplex based platform for label-free monitoring of DNA reaction kinetics. Analyst 143, 1444–1453 (2014).
19. Nie, J., Zhang, D. W., Tie, C., Zhou, Y. L. & Zhang, X. X. G-quadruplex based two-stage isothermal exponential amplification reaction for label-free DNA colorimetric detection. Biosens. Bioelectron. 56, 237–242 (2014).
20. Liao, A. M. et al. A Simple Colorimetric System for Detecting Target Antigens by a Three-Stage Signal Transformation-Amplification Strategy. Biochemistry 57, 5117–5126 (2018).
21. Li, W. et al. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: Adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity. Nucleic Acids Res. 44, 7373–7384 (2016).
22. Cleland, W. W. Dithiothreitol, a New Protective Reagent for SH Groups. Biochemistry 3, 480–2 (1964).
23. Lukesh, J. C., Palte, M. J. & Raines, R. T. A potent, versatile disulfide-reducing agent from aspartic acid. J. Am. Chem. Soc. 134, 4057–4059 (2012).
24. [http://parts.igem.org/Part:BBa\_K1614007](http://parts.igem.org/Part%3ABBa_K1614007)
25. Chakrabarti, R. & Schutt, C. E. The enhancement of PCR amplification by low molecular-weight sulfones. Gene 274, 293–8 (2001).
26. Hardjasa, A., Ling, M., Ma, K. & Yu, H. Investigating the Effects of DMSO on PCR Fidelity Using a Restriction Digest-Based Method. J. Exp. Microbiol. Immunol. 14, 161–164 (2010).
27. Nakano, S. ichi & Sugimoto, N. The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents. Biophysical Reviews 8, 11–23 (2016).

### About Us

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# Team:Leiden/Experiments

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iGem Leiden 2020

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## Rapidemic

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# Experiments

![](https://static.igem.org/mediawiki/2020/2/27/T--Leiden--LogoScience.png)

With the limited space available in the Dutch laboratories due to COVID-19, we only had four weeks to perform our experiments and prove a working mechanism for our diagnostic test. Nevertheless, we had four successful weeks in the lab, during which we managed to demonstrate a proof-of-concept for the technology. The results have formed a key part in the development of our project. Below, you can find an overview of the experiments that we performed and their corresponding protocols.

Experiments

Protocols

RPA

LSDA

GQ-catalyzed Oxidation

References

RPA

LSDA

GQ-catalyzed Oxidation

Our nucleic acid detection method consists of three reactions: recombinase polymerase amplification (RPA), linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ)-catalyzed oxidation. Further down this page we explain how these reactions work. First, we characterized each reaction individually. Then, once we knew the individual parts were successful on their own, we could combine them to achieve the proof of concept of the complete mechanism.

Prior to the time in the lab, we prepared the initial protocols for each experiment. However, during the experiments, we had to adjust our initial protocols to optimize the individual reactions to our preferences or to get them to work in a combined manner. Below, we describe the overall goals of the three main reactions of our project, and for each, you can find the optimized protocol. Changes to protocols for certain experiments are referred to in the [Results](https://2020.igem.org/Team%3ALeiden/Results) section.

## Recombinase polymerase amplification (RPA)

RPA is a promising isothermal amplification method for versatile point-of-care applications due to its low reaction temperature and relatively simple primer design1,2. During the reaction, a recombinase assists the binding of two primers to the template DNA upon which a strand-displacement polymerase amplifies the target sequence1. We designed RPA primers to target specific sequences on (synthetic) DNA from *Saccharomyces cerevisiae, Bacillus subtilis, Plasmodium falciparum, Mycobacterium sp.* and influenza A H1N1 using PrimedRPA3. This collection of targets was chosen because of its variety in types of microorganisms (yeast, bacteria, parasite, virus). By demonstrating that the RPA reaction can be applied to detect not only few, but various types of micro-organisms, we show the versatility of our detection method. In addition, while the pathogenic *Plasmodium falciparum, Mycobacterium sp.* and influenza A virus only allow to work with small synthetic parts due to safety reasons, *Saccharomyces cerevisiae* and *Bacillus subtilis* are harmless and allow to work with full genomes.

The designed target and primer sequences can be found in [Table 2 and 3 on the Parts](https://2020.igem.org/Team%3ALeiden/Parts) page. The reverse primers have an overhang of ~27 nucleotides that contains the reverse complementary sequence of recognition site for a nicking endonuclease (nickase) and a GQ sequence, which is needed to couple RPA with the subsequent SDA reaction (Fig. 1).

First, experiments were performed to test if RPA can be performed with our designed primers. Then, experiments followed to obtain the temperature-dependency and the kinetic rate of the reactions. Lastly, we tried to determine the lower limit of detection. For most of the experiments, we used genomic DNA isolated from Saccharomyces cerevisiae strain BY4741. Additionally, with the amplification of small synthetic DNA from *S. cerevisiae, Bacillus subtilis, Plasmodium faciparum, Mycobacterium sp.*  and influenza A H1N1, we intended to show the versatility of our detection method; we showed that the RPA reaction can be applied to detect not only few, but various target organisms.

Click here to read the protocol.

![](https://static.igem.org/mediawiki/2020/c/cc/T--Leiden--Experiments_figure1_RPA.png)

**Fig. 1 RPA with a non-tailed forward primer and a tailed reverse primer.**  The tail of the reverse primer contains the reverse complementary sequences of a nickase recognition site (N') and a GQ sequence (G'). As a result, the top strand of the amplification product contains a nickase recognition site (N) and a GQ sequence (GQ).

## Linear strand-displacement amplification (LSDA)

In the linear strand displacement amplification (LSDA) reaction, a nickase makes a single-stranded cut in the double-stranded RPA product (Fig. 2)4. Then, a strand-displacement polymerase starts elongating the 3' end of the DNA at the site of the cut, releasing the GQ sequence from the RPA product.

Experiments characterizing LSDA were performed to determine which nickases work and at which conditions. The nickases used were Nt.AlwI, Nt.BsmAI and Nt.BstNBI. In addition, we varied the concentrations of the nickase and reaction volumes to see if we can scale down the reaction to reduce the costs of the diagnostic test in a later stage.

Click here to go to the protocol.

![](https://static.igem.org/mediawiki/2020/f/f7/T--Leiden--LSDA-animation.gif)

**Fig. 2 Linear strand-displacement amplification (LSDA) mechanism.**  The nickase binds at its recognition site in the RPA product and makes a single-stranded cut in the top strand of the double-stranded DNA complex. Then, a strand-displacement DNA polymerase binds at the cut site and elongates the top strand starting from the 3' end.

## GQ-catalyzed oxidation

In the oxidation reaction, a sequence of single-stranded DNA forms a guanine-quadruplex (GQ) structure, stabilized by potassium ions and hemin (Fig. 3a). This structure has a peroxidase-mimicking activity; the DNAzyme can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2) to produce a color change5, 6 (Fig. 3b). The oxidation reaction has two reaction products with distinct colors. During the reaction, we analyzed the formation of the first reaction product (blue) with the absorbance at 370 and 650 nm. Then, we added a strong acid to push the reaction to the second reaction product (yellow), and measured the absorbance at 450 nm.

First, GQ oxidation experiments were performed to examine the activity of three different DNAzymes: [BBa\_K3343000](http://parts.igem.org/Part%3ABBa_K3343000), [BBa\_K1614007](http://parts.igem.org/Part%3ABBa_K1614007) and [BBa\_K3343001](http://parts.igem.org/Part%3ABBa_K3343001). ([Parts](https://2020.igem.org/Team%3ALeiden/Parts).) Then, we characterized the most potent DNAzyme of the three, BBa\_K3343000, more extensively to obtain its kinetic parameters and activity in different buffers. The GQ oxidation reaction was also used to analyze LSDA reactions and later to demonstrate the complete proof-of-concept of our detection mechanism.

Click here to go to the protocol.

![](https://static.igem.org/mediawiki/2020/6/67/T--Leiden--Engineering8_Experiments_GQ-Oxidation.jpg)

**Fig. 3 Guanine-quadruplex (GQ)-catalyzed TMB oxidation. a)**  Single-stranded GQ sequences form a GQ structure with peroxidase-mimicking activity upon binding potassium ions and hemin. **b)** The GQ DNAzyme catalyzes the oxidation of TMB in the presence of hydrogen peroxide (H2O2). First, an intermediate product, a radical cation-TMB/diimine complex, is formed with a blue color. The addition of sulfuric acid then pushes the TMB oxidation reaction to the second reaction product, a diimine with yellow color.

[Protocols](#marker)

## References

1. Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA Detection Using
   Recombination Proteins. PLoS Biol. 4, e204 (2006).
2. Zanoli, L. & Spoto, G. Isothermal Amplification Methods for the Detection of Nucleic
   Acids in Microfluidic Devices. Biosensors 3, 18–43 (2012).
3. Higgins, M. et al. PrimedRPA: primer design for recombinase polymerase
   amplification assays. Bioinformatics 35, 682–684 (2019). <https://doi.org/10.1093/bioinformatics/bty701>
4. Joneja, A. & Huang, X. Linear nicking endonuclease-mediated strand-displacement
   DNA amplification. Anal. Biochem. 414, 58–69 (2011).
5. Nie, J., Zhang, D.-W., Tie, C., Zhou, Y.-L. & Zhang, X.-X. G-quadruplex based two-stage
   isothermal exponential amplification reaction for label-free DNA colorimetric
   detection. Biosens. Bioelectron. 56, 237–242 (2014).
6. Li, W. et al. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine
   as the intramolecular species for remarkable enhancement of enzymatic activity.
   Nucleic Acids Res. 44, 7373–7384 (2016).

RPA

*Text adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08)*

Click here to return to the experiment.

## Genetic sequences, enzymes, and reagents

Primers were purchased from Sigma-Aldrich. Purified Saccharomyces cerevisiae BY4741 genomic DNA was kindly obtained from Paul van Heusden (Leiden, Netherlands). TwistAmp Basic RPA kit was purchased from TwistDx.

## Standard RPA reaction

The RPA reaction mix was assembled in a total volume of 47.5 µL, with

* 29.5 µL Rehydration buffer (provided with the TwistAmp Basic kit);
* 2.4 µL of each primer (10 µM);
* 5 µL template; and
* nuclease-free water.

The pellet (provided with the TwistAmp Basic kit) was rehydrated by adding 47.5 µL RPA reaction mix and was incubated for 5 minutes until completely dissolved. The reaction mix was mixed by pipetting. The reaction mix was divided into 19-µL aliquots. To start the reaction, 1 µL of magnesium acetate solution (provided with the TwistAmp Basic kit) was added. The reaction was incubated at 42 °C for 20 minutes. The reaction was terminated by quenching the reaction on ice or by PCR clean-up when the amplification products were subsequently analyzed by gel electrophoresis.

### Analysis

After following a routine clean-up using PCR clean-up kit (QIAGEN), amplification was confirmed by agarose gel electrophoresis (2% agarose gel) and Nanodrop.

## Real-time RPA

2.5 µL of 20x SYBR green dye was added to a 50 µL RPA mixture. Before starting the amplification reaction with MgOAc solution, the whole RPA mixture was transferred to a 96-well RT-PCR plate. The BioRad CFX96 Touch Real-Time PCR Detection system was used to measure the signal every 30 seconds at 42 °C.

The measurements enclosed in the file named 'Quantification Amplification Results' are used to make a graph which visualizes the amplification of the target over time. When separate names are used for each of the samples, the file named 'Melt curve plate view results' can be used to determine whether no other targets were amplified. This is especially useful when the entire genomes are used as templates.

Click here to return to the experiment.

LSDA

*Text adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08)*

Click here to return to the experiment.

## Genetic sequences, enzymes, and reagents

Synthetic oligo DNA as LSDA templates were purchased from BaseClear (Leiden, Netherlands). Nicking endonucleases, including Nt.BstNBI, Nt.AlwI and Nt.BsmAI, and Bst 2.0 DNA Polymerase were acquired from New England Biolabs (NEB). dNTP Mix was purchased from Merck.

## LSDA reaction

The LSDA reaction mix was assembled on ice in a total volume of 10 µL, with

* 1 mM dNTP Mix;
* 0.5X NEBuffer 3.1;
* 0.5X Isothermal Amplification Buffer;
* 0.4 U Bst 2.0 DNA Polymerase;
* 3.2 U nickase (Nt.AlwI, Nt.BsmAI, Nt.BspQI or Nt.BstNBI);
* 1 µL template; and
* nuclease-free water.

Reaction mix was prepared in bulk and divided into 10-µL aliquots. The LSDA reaction mix was incubated at 55 °C for 40 minutes. The reaction was quenched on ice.

## LSDA reaction for initial characterization of nickases

LSDA reactions for initial characterization of nickases were performed with synthetic templates (1.6 x 1010 copies/µL) to eliminate noise from the RPA reaction. Double Bst 2.0 Polymerase and nickase concentrations were used to accommodate signal production from the low template concentration.

LSDA reaction mix was assembled on ice in a total volume of 10 µL, with

* 1 mM dNTP Mix;
* 0.5X NEBuffer 3.1;
* 0.5X Isothermal Amplification Buffer;
* 0.8 U Bst 2.0 DNA Polymerase;
* 6.4 U nickase
* 4 µL template; and
* nuclease-free water.

Reaction mix was prepared in bulk and divided into 10-µL aliquods. The LSDA reaction mix was incubated for 40 minutes at 55 °C for Nt.BstNBI and 50 °C for Nt.AlwI and Nt.BsmAI. Reactions were quenched by placing the tubes on ice.

### Analysis

The LSDA reaction was analyzed using GQ oxidation.

Click here to return to the experiment.

GQ-catalyzed Oxidation

*Text adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08)*

Click here to return to the experiment.

## Genetic sequences, enzymes, and reagents

DNAzymes were purchased from Sigma-Aldrich. Chemical reagents were purchased from Merck.

## GQ oxidation reaction

0.1 M phosphate citrate buffer was assembled at pH 3.8 with

* 0.071 M Na2HPO4.7H2O (MW 268.07 g/mol);
* 0.0645 M citric acid; and
* HCl or NaOH to adjust to desired pH.

Hemin and TMB were dissolved in DMSO.
KCl and H2O2 were dissolved in phosphate citrate buffer pH 3.8 (no DMSO).

Hemin/KCl solution was assembled with:

* 1.165 mg/mL KCl;
* 2.5 µM hemin; and
* phosphate citrate buffer pH 3.8.

TMB/H2O2 solution was assembled with

* 0.12 mg/mL TMB;
* 0.09% (w/w) H2O2; and
* phosphate citrate buffer pH 3.8.

The wells of a 96-well plate were filled with 1.25 µL LSDA product, 0.875 µL phosphate citrate buffer (pH 3.8) and 40 µL hemin/KCl solution. LSDA product and phosphate citrate buffer were replaced with 10 µL 1 µM EAD2+3'A (DNAzyme, 5’ CTGGGAGGGAGGGAGGGA) or 10 µL phosphate citrate buffer for positive and negative controls, respectively. To start the reaction, 50 µL of freshly prepared TMB/H2O2 start solution was added. To stop the reaction, 50 µL 0.5 M sulfuric acid was added.

The 96-well plate (filled with hemin/KCl solution and LSDA product) and start solution were incubated for 10 minutes at 25 °C before the reaction was started.

### Analysis

Using the SparkTM 10M multimode microplate reader, the absorbance at 370, 450 and 650 nm was measured every 40 seconds for 30 minutes. After stop solution was added, the absorbance at 450 nm was measured once again. The plate reader was pre-heated at 25 °C.

Click here to return to the experiment.

[Experiments](#marker)

### About Us

We are the 2020 team of iGEM Leiden. With an interdisciplinary team of students we aim to develop a point-of-care rapid diagnostic tool for infectious diseases!

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# Team:Leiden/Results

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iGem Leiden 2020

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## Rapidemic

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# Results

![](https://static.igem.org/mediawiki/2020/2/27/T--Leiden--LogoScience.png)

Once the mechanism of our detection technique was designed, we performed laboratory experiments to determine the experimental conditions of the new technology. After a few rounds of optimization, a proof-of-concept experiment showed how the reactions of the technology can be successfully connected to detect, amplify and report the presence of a target DNA. On this page, we show the experiments that led to the proof-of-concept in detail. The results were also published as a pre-print on BioRxiv; the complete publication can be viewed after the Discussion section.

Overview

The reactions

Coupling the reactions

Discussion

Introduction

Individual characterization of each reaction

Proof-of-Concept: Coupling the Reactions

Future Prospects

References

RPA

LSDA

Oxidation

References

DTT inhibits GQ oxidation but is inactivated at low pH

Coupling RPA, LSDA, and TMB oxidation

DMSO reduced primer dimer formation during RPA and improved method accuracy

References

Discussion

Publication

References

## Introduction

Recently, a novel severe acute respiratory syndrome (SARS), coronavirus disease 2019 (COVID-19), spread worldwide, causing over 27,800,000 cases of infection and 900,000 deaths as of September 10th, 20201. Although this was the most recent pandemic, it was not the first outbreak that caused major health issues on a global scale, and it will also not be the last one. Potential future epidemic-causing pathogens are listed in the WHO R&D Blueprint and also include unknown diseases, collectively named “Disease X”2. To be prepared for "Disease X" (and other future infectious diseases), we developed an innovative detection technique, called Rapidemic, that can be rapidly adaptable to new pathogens. This technique is based on an isothermal amplification method coupled to a colorimetric readout for nucleic acid detection. Due to its ability to be used in point-of-care settings, it has the potential to enable population testing worldwide.

The assay is built on three reactions to yield a color change visible to the naked eye (Fig. 1). The working mechanisms of these reactions are explained in detail on the Engineering page. In short, the first reaction is recombinase polymerase amplification (RPA). RPA is an amplification reaction that works at a low and constant temperature, during which a specific target sequence is exponentially amplified. The product of this reaction serves as a template for the second reaction of the assay, the linear strand-displacement amplification (LSDA) reaction. Here, a short single-stranded DNA sequence, a guanine-quadruplex (GQ) DNAzyme, is produced. During the last reaction of the assay, the DNAzymes catalyze an oxidation reaction to produce the color change. To validate the new detection technique, we first characterized each of the reactions individually. This was followed by a proof-of-concept study during which we coupled the three reactions.

![](https://static.igem.org/mediawiki/2020/1/1e/T--Leiden--Results1_Hemin-GQ-min.png)

**Fig. 1 Amplification scheme of the DNAzyme-based label-free detection method Rapidemic.** The Rapidemic detection method relies on three main reactions: RPA, LSDA, and GQ-hemin DNAzyme-catalyzed TMB oxidation. During RPA, the target sequence is amplified and extended with a short segment of nucleotides due to primer overhang. This segment contains the reverse complementary sequences of a recognition site for a nicking endonuclease (nickase) and a GQ sequence. The nickase makes a single-stranded cut near its recognition site in the amplification product, after which a strand-displacement polymerase elongates the 3’ end at the nickase cut site and thereby displaces the single-stranded GQ sequence, so that it is released from the double-stranded DNA molecule. The single-stranded GQ sequence forms a three-dimensional structure with peroxidase activity upon binding to potassium ions and hemin. Finally, the DNAzyme catalyzes the oxidation of 3,3′,5,5′-tetramethylbenzidine sulphate (TMB) in the presence of hydrogen peroxide (H2O2) to produce a color change. As a proof-of-concept for this principle, we reported the initial validation of this detection method with genomic DNA of *S. cerevisiae.* Figure adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08).

## Individual characterization of each reaction

### First reaction: RPA

The RPA characterization experiments were aimed to:

+ Confirm amplification with designed primers
+ Study RPA kinetics at different temperatures
+ Scale down RPA reaction volume
? Determine RPA detection limit
? Resolve false-positive primer-associated amplification

(+ and ? indicate "successful" or "remains a challenge")

For most RPA experiments, we used isolated genomic DNA of *Saccharomyces cerevisiae (S. cerevisiae)* as the amplification target. We performed RPA using the commercial TwistAmp Basic RPA kit (TwistDx). To target a specific sequence on the genome of *S. cerevisiae*, we designed a set of primers using a freely available software program called PrimedRPA3. The primer sequences can be found in Table 3 on the [Parts](https://2020.igem.org/Team%3ALeiden/Parts) page. To couple the RPA reaction to the second reaction in our detection scheme (LSDA), we inserted a 27-bp overhang sequence at the 5’-end of the reverse primer. This overhang sequence contained the reverse complementary sequence of a nickase recognition site and the EAD2+3’A DNAzyme, separated by a short spacer ( <10bp).

The main results of our RPA experiments were:

1. The RPA primer pairs were able to successfully amplify their target sequence (Fig. 2).
2. The presence of 5’-overhang (reverse primer) did not interfere with the amplification of the target sequence (Fig. 2).
3. The lowest temperature limit for RPA was 20 °C (Fig. 3).
4. For > 10 minutes incubation time, the amplification yield does not depend on the incubation temperature in the range of (at least) 30 - 42 °C. For incubation time < 10 minutes, there is a tradeoff between high yield and low temperature (Fig. 4).
5. The final RPA reaction volume is 20 µL.

At first, when we performed RPA reactions with a very low concentration of target DNA to test the sensitivity of RPA, the gel electrophoresis and real-time RPA results showed false-positive signals. The off-target amplification presumably caused the inconsistent results that we observed in these experiments (Fig. S2), and they were presumably caused by primer-associated amplification initiated through primer secondary structures and/or primer-primer interactions (Fig. 10b). The addition of 2.5% DMSO as an amplification enhancer to the RPA reaction successfully reduced the false-positive signals for *S. cerevisiae*, as analyzed by real-time RPA using the SYBR Green dye (Fig. 10c).

We then performed RPA reactions with small synthetic templates derived from various other microorganisms to show the versatility of our detection system. These targets organisms were *S. cerevisiae, Bacillus subtilis, Plasmodium falciparum, Mycobacterium sp.* and influenza A H1N1. However, only for *S. cerevisiae* and influenza A, amplifications were obtained without a false-positive signal when analyzed by real-time RPA using the SYBR Green dye (Fig. S3).

### Second reaction: LSDA

The LSDA characterization experiments were aimed to perform the following:

+ Compare different nickases and identify the most efficient one
+ Reduce the nickase concentration and the LSDA reaction volume
? Study the reaction kinetics at different temperatures

The LSDA reaction is the bridge linking the first amplification step (RPA) to the signal reporting step (TMB oxidation). LSDA uses a nickase to produce specific single-stranded DNA sequences, namely GQ DNAzymes. For this, the nickase requires a template DNA molecule, which is the product of the RPA reaction. During the LSDA characterization experiments, we aimed to identify the performance of different nickases (Nt.BstNBI, Nt.AlwI, and Nt.BsmAI), for which we used synthetic templates (see Table 2 on the [Parts](https://2020.igem.org/Team%3ALeiden/Parts) page). Since the nickases are one of the most expensive components of our detection method (Fig. 3 on [Proof of Concept](https://2020.igem.org/Team%3ALeiden/Proof_Of_Concept) page), we also aimed to optimize the nickase concentration and LSDA reaction volume. For that, we used self-generated RPA products.

The main results from these experiments were:

1. The Nt.BstNBI was the most efficient nicking endonuclease (Fig. 5).
2. The use of Nt.BstNBI was decreased 40-fold by reducing the nickase concentration 8-fold (Fig. S4) and the LSDA reaction volume 5-fold (Fig. S5).

We did not manage to study the LSDA kinetic rates at different temperatures due to a lack of time. We planned to use the kinetic parameters in our (semi-)empirical [model](https://2020.igem.org/Team%3ALeiden/Model) that simulates the underlying reaction kinetics of our detection method. Instead, we adopted kinetic parameters from literature to build the model.

### Third reaction: DNAzyme-catalyzed oxidation

The DNAzyme-catalyzed oxidation experiments were aimed to perform the following:

+ Confirm functioning DNAzyme (New Part added to iGEM Registry and Existing Part improved)
+ Determine kinetic parameters of DNAzyme to incorporate into [model](https://2020.igem.org/Team%3ALeiden/Model)
? DNAzyme concentration calibration curve with optimized reaction conditions

The last reaction of our detection technique is the GQ DNAzyme-catalyzed TMB oxidation reaction. GQ-hemin complexes catalyze the oxidation reaction of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide, resulting in a color change. As the color change is observable by the naked eye, we can enable the detection of pathogenic genetic material in a sample without the requirements for complex laboratory equipment. We tested a variety of GQ DNAzymes to select the most efficient one. We also varied the concentration of the substrate (TMB) to obtain the kinetic parameters of the most efficient DNAzyme.

The main results from these experiments were:

1. The most potent GQ DNAzyme was EAD2+3’A (Fig. 6).
2. The addition of an adenine base at the 3'-end of DNAzyme BBa\_K1614007 greatly boosted its activity (Fig. 6).
3. The molar maximum TMB conversion rate (kcat) of EAD2+3'A was 3.971x104 min-1 M(DNAzyme)-1, and the TMB affinity of EAD2+3’A was 6.798x10-3 g L-1 (Fig. 7 and Table 1).

[Click here](#marker) to go to the Results section of the individual characterization of the three reactions.

## Proof-of-concept: coupling the reactions

The proof-of-concept experiments were aimed to perform the following:

+ Couple RPA, LSDA and GQ-catalyzed oxidation in a serial manner
? Combine RPA and LSDA in a one-pot reaction
? Determine the detection limit of the detection method
? Optimize reactions for low false-positive and false-negative rates

Having optimized each reaction separately, we aimed to couple RPA, LSDA and TMB oxidation reactions and validate our assay. Therefore, we passed the product of each preceding reaction to the next in a serial manner. Initially, we did not succeed in coupling all three reactions; no positive colorimetric signal from GQ-hemin catalyzed TMB oxidation was detected. Therefore, we started to investigate possibilities for optimization of the coupling step.

Based on the optimization steps we performed we could conclude the following:

1. Purification of the RPA product prior to the LSDA reaction resulted in a colorimetric signal (Fig. S7); a compound in the RPA mix interfered with the oxidation reaction.
2. The reducing agent DTT present in the RPA mix inhibited the oxidation reaction (Fig. S8).
3. Lowering the pH of the oxidation buffer decreased the inhibiting effect of DTT on the oxidation (Fig. 8).
4. Lowering the pH of the oxidation buffer was not sufficient to enable color generation; the amount of RPA product in the oxidation reaction was too high (Fig. 9a and b).
5. Optimization of the dilution rates of the RPA and LSDA products enabled color generation (Fig. 9c and d).
6. False-positive readouts were generated as a consequence of primer self-amplification and/or primer-primer interactions (Fig. 10a and b).
7. The addition of 2.5% DMSO to the RPA mix showed to be an efficient strategy to reduce false-positive signal (Fig. 10c and d).

[Click here](#marker) to go to the Results section of the coupling of the three reactions.

## Future Prospects

*Text adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08)*

Here we presented a novel design of a label-free DNAzyme-based detection method called Rapidemic. This assay combined RPA with linear strand-displacement amplification (LSDA) and guanine-quadruplex (GQ) DNAzyme-catalyzed color-changing reaction. The colorimetry basis of the signal readout omitted the need for extensive instrumentation. Moreover, primer-based sequence detection of RPA gave Rapidemic a potential to be rapidly adapted to target a new sequence. As a proof of concept, we developed the assay to detect isolated genomic DNA of Saccharomyces cerevisiae. The use of low-pH buffers and the optimization of the dilution rates from each preceding reaction to the next showed to be successful strategies to enable visible detection with this method. These findings demonstrate for the first time that a label-free DNAzyme-based detection method can be coupled to RPA and LSDA for nucleic acid detection.

Despite successful integration of the three reactions, the technique presented significant false-positive signals in the absence of target sequence. Although the addition of DMSO in the RPA reaction effectively reduced the occurrence of false-positive signals, the less distinct color change due to DMSO revealed the need to further improve and optimize the presented sequence detection method to be used as a rapid diagnostic tool. Future work may focus on alternative enhancing chemicals that do not interfere with the oxidation reaction, as well as improved primer sequences that are less prone to have off-target interactions.

Despite the need for further optimization, the presented sequence detection method holds potential to be a rapid diagnostic tool. Its RPA-based detection allows it to be repurposed to target other DNA sequence by simply changing its primers, similar to the development of PCR-based tests. This allows rapid adaptation of the method to target newly emerging diseases. In comparison, development of antibody and/or antigen-based tests may require significantly more time and resources to be redeveloped to target a new disease4. Additionally, [the reagent costs of the presented detection method are similar to commercial PCR-based tests](https://2020.igem.org/Team%3ALeiden/Proof_Of_Concept). However, unlike PCR-based tests, our method does not require extensive instrumentation to give a signal readout. Altogether, the method presents a potential to be a rapidly adaptable point-of-care diagnostic tool.

Future research may focus on combining RPA and LSDA into a one-pot reaction. The development of a one-pot reaction may further hasten the reactions’ incubation period and reduce the reagent costs (dNTPs, buffers, polymerase). Moreover, this would greatly simplify the hardware design when applied in point-of-care diagnostics. It may be necessary to replace the commercial TwistDx RPA kit with a custom-made RPA mix to optimize its composition for one-pot RPA and LSDA. With such custom-made mix, the concentration of DTT could be reduced to increase the amount of RPA product allowed in the oxidation reaction and increase the method’s sensitivity. Altogether, the presented sequence detection scheme presented a promising potency as rapid point-of-care diagnostic tool that may contribute in future attempts to diagnose, and by doing so, monitor and contain the spread of an infectious disease.

Click here to read the full discussion.

Visit our [Proof of concept](https://2020.igem.org/Team%3ALeiden/Proof_Of_Concept) to see how we integrated our successful detection method into a preliminary prototype device.

[The reactions](#marker)

## References

1. John Hopkins University and Medicine. COVID-19 map. John Hopkins Coronavirus Resour. Cent. (2020). Available at: <https://coronavirus.jhu.edu/map.html.> (Accessed: 10th September 2020).
2. World Health Organization. WHO R&D Blueprint: list of blueprint priority diseases. (2020). Available at: [http://www.who.int/blueprint/priority-diseases/en.](http://www.who.int/blueprint/priority-diseases/en.%20)(Accessed: 11th September 2020).
3. Higgins, M., Ravenhall, M., Ward, D., Phelan, J., Ibrahim, A., Forrest, M. S., Clark, T. G., and Campino, S. (2019). PrimedRPA: primer design for recombinase polymerase amplification assays. Bioinformatics, 35(4), 682–684. [https://doi.org/10.1093/bioinformatics/bty701.](https://doi.org/10.1093/bioinformatics/bty701)
4. Peeling, R. W., Murtagh, M. & Olliaro, P. L. Epidemic preparedness: why is there a need to accelerate the development of diagnostics? The Lancet Infectious Diseases 19, 172–178 (2019).

## RPA

### Confirmation of target amplification using recombinase polymerase amplification (RPA)

*Text and figure adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08)*

Our method employed an initial target amplification step. This step is essential to selectively amplify the positive signal from the targeted sequence(s) present in the sample. Initial target amplification was performed using the TwistAmp Basic RPA kit (TwistDx). The primer pairs used in this experiment were designed to target a 191 bp sequence from the genome of *Saccharomyces cerevisiae (S. cerevisiae)*. In addition to the target-specific sequences, the reverse primer contained a 27 bp overhang sequence at its 5’-end. This primer overhang encoded for a nickase recognition site, a short spacer (less than 10 bp), and a sequence reverse complementary to EAD2+3’A DNAzyme. Here, we evaluated the primer pairs’ ability to detect the presence of its target sequence by using *S. cerevisiae* BY4741 genome as target sample (Table S2). As can be seen from Fig. 2, the inclusion of 5’-overhang sequence did not appear to have interfered with RPA’s ability to detect the presence of its target sequence.

Primer sequences can be found in Table 3 on the [Parts](https://2020.igem.org/Team%3ALeiden/Parts) page.

![](https://static.igem.org/mediawiki/2020/4/47/T--Leiden--Results2_RPA.png)

**Fig. 2 RPA with forward primer SC1\_F\_p1 and reverse primers SC1\_R\_Nt.AlwI\_EAD2+3A (lane 2 and 3) or SC1\_R\_Nt.**BstNBI\_EAD2+3A (lane 4 and 5) targeting *S. cerevisiae* genome. Each reaction was performed using genomic *S. cerevisiae* BY4741 DNA as a target sequence. The target sequence is absent in the negative control. The positive control contains the primers and target templates provided by the TwistAmp Basic kit (TwistDx).

Read More
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One of the two samples with reverse primer with the Nt.BstNBI recognition site did not work in the previous experiment (Fig. 2). This may have been caused by an experimental error. Therefore, we repeated the experiment, including additional samples with reverse primers with the recognition site for Nt.BsmAI or Nt.BspQI. This time, bands were observed at the expected height for all replicates for the Nt.AlwI and Nt.BstNBI primer sets (Fig. S1). For the Nt.BsmAI primer set, no amplification was observed. For the Nt.BspQI primer set, only one of the two replicates showed a band at the expected height. We continued our experiments with the Nt.BstNBI and Nt.AlwI primer sets.

![More resutls](https://static.igem.org/mediawiki/2020/a/a7/T--Leiden--Results3_RPA.png)

**Fig. S1 RPA with forward primer (SC1\_F\_p1) and reverse primers (SC1\_R\_Nt.AlwI\_EAD2+3A or SC1\_R\_Nt.BstNBI\_EAD2+3A or SC1\_R\_Nt.BsmAI\_EAD2+3A or SC1\_R\_Nt.BspQI\_EAD2+3A) targeting *S. cerevisiae* BY4741 genome.**  The reactions were performed in duplicate.

### RPA kinetics at different temperatures

The recommended temperature for RPA reactions performed with the TwistAmp Basic RPA kit (TwistDx) is 37 to 42 °C1. However, performance at lower temperatures may be advantageous when the reaction is integrated for point-of-care use. Previous studies showed that RPA can work while it is heated by holding it in the palm of the hand2. Performing RPA at room or body temperature will significantly simplify the design of a diagnostic kit as the incorporation of a heating instrument would no longer be required.

As both incubation time and temperature affect the performance of RPA, several experiments were performed to test both variables in tandem. RPA reactions were performed at five different temperatures (4, 20, 25, 30 and 42 °C). Four identical samples were incubated in a PCR machine at each temperature. At intervals of 5 minutes, one sample was taken out of the PCR machine and placed on ice.

Fig. 3 confirms the Amplification of the expected target sequence was confirmed for all tested temperatures (Fig. 3). The samples incubated at 4 °C served as negative controls, and as expected no band was observed. For 20 °C, a minimum incubation period of 15 minutes was needed to confirm amplification of the target on the gel. However, for 42 °C, 5 minutes of incubation was enough to confirm amplification. When we compare the samples incubated for 20 minutes at different temperatures, we can observe that the highest yield is at 30 ºC. Thus, this experiment showed that RPA can work at temperatures as low as 20 °C, although the yield at 20 °C was significantly lower than for 25 °C to 42 °C. Further experiments are needed to show whether the amplification yield at 20 °C is sufficient for sensitive detection with our detection method (RPA coupled to LSDA and DNAzyme-catalyzed oxidation).

![Results](https://static.igem.org/mediawiki/2020/9/90/T--Leiden--Results4_RPA_kinetics-min.png)

**Fig. 3 RPA kinetics at different incubation temperatures.** Forward primer (SC1\_F\_p1) and reverse primer (SC1\_R\_Nt.AlwI\_EAD2+3A) targeted *S. cerevisiae* BY4741 genome. Positive and negative controls were incubated for 20 minutes. Amplification was not confirmed for the positive control at 25 °C, which could mean that this specific primer set (supplied with the TwistDx RPA kit) does not work at this temperature, or it could be due to an experimental error. No amplification appears to occur at 4 °C, both in the sample and positive control.

In the previous experiment (Fig. 3), the incubator was opened every five minutes to take one sample out. This could have influenced the amplification process as the temperature was lowered every time the PCR machine was opened. Next, to monitor the amplification kinetics more accurately and more frequently, real-time RPA (SYBR Green dye) assays were performed at 30, 37 and 42 °C. Unfortunately, it was not possible to perform RPA at 20 °C due to time restrictions.

With real-time RPA, we confirmed the previous results that amplification can be performed at 30, 37 and 42 °C (Fig. 4). The rate of the reaction is higher at 37 °C than at 42 °C. While the reaction performed at 30 °C at some point reaches a reaction rate that is similar to the reaction performed at 37 °C, the reaction started significantly slower at 30 °C. Thus, for a reaction of 5 minutes, it is important to incubate at the optimal temperature (37 °C) to obtain a high amplification yield. In contrast, for a reaction of 10 minutes, the temperature is a less important factor as the amplification yield was similar for all temperatures at this time point. Thus, it is important to consider the tradeoff between a high amplification yield, a short reaction time and a low temperature when RPA is implemented in a point-of-care diagnostic test.

![](https://static.igem.org/mediawiki/2020/f/f2/T--Leiden--Results5_RealTime_RPA.png)

**Fig. 4 Real-time RPA with S. cerevisiae primer set and genome.** Forward primer (SC1\_F\_p1) and reverse primer (SC1\_R\_Nt.BstNBI\_EAD2+3A) targeted S. cerevisiae BY4741 genome. Graphs were normalized by subtracting the fluorescence value at time point zero from all measurements for each temperature. Replicates: n = 3 for 30 °C and 37 °C, n = 2 for 42 °C.

### RPA sensitivity and template concentrations

To test the sensitivity of RPA, RPA reactions were performed with different template concentrations. The concentration of the template stock was approximated using Nanodrop. RPA reactions with a template concentration ranging from 108 to 10-2copies were analyzed with real-time RPA using the SYBR Green dye and gel electrophoresis. The high copy numbers were included to determine how a template overload may affect RPA.

Both the real-time RPA results and gel electrophoresis results showed inconsistencies (Fig. S2). For instance, some template concentrations clearly suggested amplification of the target sequence, while other samples with higher template concentrations showed less or no amplification. The gel electrophoresis results also indicated the occurrence of off-target amplification; bands were obtained at 100 bp and lower, a sequence length that is significantly shorter than the targeted sequence of 191 bp (Fig. S2c and d). Off-target amplification may have been caused by primer-primer interactions and/or primer secondary structures (as reinforced later in Fig. 10b). The off-target amplification may explain the inconsistent results that we obtained from these sensitivity experiments. The results from these experiments were therefore treated as inconclusive.

Supplementary figures (Fig. S2)
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![](https://static.igem.org/mediawiki/2020/c/c5/T--Leiden--Results6_RPA_sensitivity-min.png)

**Fig. S2 RPA sensitivity analysis with different template concentrations.** Forward primer SC1\_F\_p1 and reverse primer SC1\_R\_Nt.AlwI\_EAD2+3A) were used with template RPA\_temp\_SC. **a) Real time RPA.** From 1 till 8: 4.23\*106 copies decreasing with 101 until 4.23\*10-1 for 8; 9 is the negative control (no template). **b) Real time RPA.** From 1 till 4: 1106 copies to 1.106 copies in 4. No negative control. **c) RPA analyzed with gel electrophoresis.** From left to right: 1.11\*108 copies to 1.11\*11 copies represented by the final lane. **d) RPA analyzed with gel electrophoresis.** From left to right: 1106 copies to 0.0106 copies in the final lane.

![A](https://static.igem.org/mediawiki/2020/4/44/T--Leiden--M_ResultsS2a.png)
![B](https://static.igem.org/mediawiki/2020/0/0d/T--Leiden--M_ResultsS2b.png)
![C](https://static.igem.org/mediawiki/2020/7/71/T--Leiden--M_ResultsS2c.png)
![D](https://static.igem.org/mediawiki/2020/8/8c/T--Leiden--M_ResultsS2d.png)

**Fig. S2 RPA sensitivity analysis with different template concentrations.** Forward primer SC1\_F\_p1 and reverse primer SC1\_R\_Nt.AlwI\_EAD2+3A) were used with template RPA\_temp\_SC. **a) Real time RPA.** From 1 till 8: 4.23\*106 copies decreasing with 101 until 4.23\*10-1 for 8; 9 is the negative control (no template). **b) Real time RPA.** From 1 till 4: 1106 copies to 1.106 copies in 4. No negative control. **c) RPA analyzed with gel electrophoresis.** From left to right: 1.11\*108 copies to 1.11\*11 copies represented by the final lane. **d) RPA analyzed with gel electrophoresis.** From left to right: 1106 copies to 0.0106 copies in the final lane.

### RPA on various target organisms

To test the modularity of our system, we performed RPA reactions with small synthetic templates derived from different microorganisms and corresponding primer sets. Template and primer sequences can be found in Table 2 and 3 on the [Parts](https://2020.igem.org/Team%3ALeiden/Parts) page. Target microorganisms included *S. cerevisiae, Bacillus subtilis, Plasmodium falciparum* (malaria), *Mycobacterium sp.* and influenza virus A. Based on previous signals of off-target amplification (Fig. S2 and Fig. 10b), 3.5% DMSO was included in the RPA mix to enhance the selectivity of the amplification reaction by reducing primer-primer interactions and primer secondary structures.

Based on real-time RPA results, it can be surmised that the primer sets designed for S. cerevisiae and the influenza A virus allow for amplification with a low false-positive signal (Fig. S3). In contrast, the results for *Bacillus subtilis, Plasmodium falciparum* and *Mycobacterium sp.* showed a high false-positive signal. Interestingly, the real-time RPA graph of *Plasmodium falciparum* shows a false-positive signal for the negative sample but no signal increase for the positive sample. This may be the result of an experimental error, such as no addition of the SYBR Green dye.

Supplementary figures (Fig. S3)
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![](https://static.igem.org/mediawiki/2020/3/37/T--Leiden--Results7_RealTimeRPA.png)

**Fig. S3 Real time RPA with primer set and synthetic template for a)** *S. cerevisiae* (primers SC1\_F\_p1 and SC1\_R\_Nt.BstNBI\_EAD2+3A), **b**) Influenza A virus (primers IA1\_F\_p1 and IA1\_R\_NtBstNBI\_EAD2+3'A), **c)** *Bacillus subtilis* (primers BS1\_F\_p1 and BS1\_R\_Nt.BstNBI\_EAD2+3A), **d)** *Plasmodium falciparum* (primers PF1\_F\_p1 and PF1\_R\_Nt.BstNBI\_EAD2+3A) and **e)** *Mycobacterium bovis* (primers MB1\_F\_p1 and MB1\_R\_Nt.BstNBI\_EAD2+3A). Primers targeted synthetic template sequences (Table 2 on Parts page). 3.5% DMSO was included in the RPA mix.

![A](https://static.igem.org/mediawiki/2020/1/12/T--Leiden--M_ResultsS3a.png)
![B](https://static.igem.org/mediawiki/2020/5/59/T--Leiden--M_ResultsS3b.png)
![C](https://static.igem.org/mediawiki/2020/f/f7/T--Leiden--M_ResultsS3c.png)
![D](https://static.igem.org/mediawiki/2020/d/df/T--Leiden--M_ResultsS3d.png)
![E](https://static.igem.org/mediawiki/2020/2/23/T--Leiden--M_ResultsS3e.png)

**Fig. S3 Real time RPA with primer set and synthetic template for a)** *S. cerevisiae* (primers SC1\_F\_p1 and SC1\_R\_Nt.BstNBI\_EAD2+3A), **b**) Influenza A virus (primers IA1\_F\_p1 and IA1\_R\_NtBstNBI\_EAD2+3'A), **c)** *Bacillus subtilis* (primers BS1\_F\_p1 and BS1\_R\_Nt.BstNBI\_EAD2+3A), **d)** *Plasmodium falciparum* (primers PF1\_F\_p1 and PF1\_R\_Nt.BstNBI\_EAD2+3A) and **e)** *Mycobacterium bovis* (primers MB1\_F\_p1 and MB1\_R\_Nt.BstNBI\_EAD2+3A). Primers targeted synthetic template sequences (Table 2 on Parts page). 3.5% DMSO was included in the RPA mix.

### RPA scale down

Decreasing the RPA reaction volume would also allow us to lower the cost of our next experiments in the lab and our final diagnostic test, thus making it more affordable for customers. Therefore, the RPA reactions were scaled down from 50 µL to 20 µL and 10 µL to test whether this would still lead to sufficient amplicon generation. The amplicons were analyzed by gel electrophoresis and Nanodrop. The amplification products for 10 and 20 µL reaction volume showed similar results as for 50 µL reaction volume (data not shown). These results suggested that RPA can be scaled down by 2.5-fold and even 5-fold. We continue our experiments with a reaction volume of 20 µL, since this method is less prone to pipetting errors than with a reaction volume of 10 µL.

## LSDA

### Confirmation of GQ DNAzyme generation with LSDA with different nickases

*Text and figure adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08).*

![](https://static.igem.org/mediawiki/2020/d/d2/T--Leiden--Result8_DNAzyme_Production.png)

**Fig. 5 GQ DNAzyme production using SDLA reaction.** SDLA was performed using different nickases. Here we identified Nt.BstNBI as the most potent GQ-producer. Synthetic (pure) oligonucleotide containing the targeted S. cerevisiae genome, nickase site, and EAD2+3’A DNAzyme sequences was used as template for the SDLA reaction.

In our sequence detection scheme, strand-displacement linear amplification (SDLA) acts as a connecting reaction to bridge initial signal generation by RPA and subsequent signal reporting by TMB oxidation. SDLA achieved this by producing GQ DNAzymes using purified RPA amplicons as templates. Here, we confirmed the effectivity of SDLA in guanine-quadruplex (GQ) sequence generation by using a synthetic (pure) template that resembles the RPA amplicon. The effectivity of Nt.BstNBI, Nt.Alwi, and Nt.BsmAI in mediating SDLA reactions was investigated. Two blank controls, which resembled the buffer conditions used in the three SDLA reactions, were included as standard of comparison. In the current reaction mix, we identified Nt.BstNBI as the most potent nickase for the production of GQ DNAzyme in an SDLA reaction (Fig. 5). This result confirmed the effectivity of the SDLA reaction in producing single-stranded GQ DNAzyme as well as its role as a bridge between RPA and TMB oxidation.

### Scale down LSDA reaction

The most expensive part of the kit was initially estimated to be the commercially available nickase, which enables amplification of the GQ sequence in the LSDA reaction. To keep the cost of our diagnostic test at a minimum, it is essential that we reduce the amount of nickase in the reaction. Therefore, we performed two experiments to test if (1) the concentration of nickase can be reduced and (2) the reaction volume can be scaled down. The concentration of nickase in the LSDA reaction could be reduced from 1.28 U/µL to at least 0.16 U/µL (8-fold) (Fig. S4), and the reaction volume can be scaled down from 25 µL to 5 µL (5-fold) (Fig. S5). This means that the amount of nickase and thus the cost for the nickase in the test kit could be reduced 40-fold.

Supplementary figures (Fig. S4-5)
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![](https://static.igem.org/mediawiki/2020/2/23/T--Leiden--9_LSDA_reactions-min.png)

**Fig. S4 LSDA reactions with different concentrations of nickase (Nt.BstNBI).** The nickase concentration was varied to 0.16, 0.32 and 0.64 U/µL. The LSDA reaction with 0.16 U/µL nickase could still give significant color change.

![](https://static.igem.org/mediawiki/2020/d/d6/T--Leiden--Results10_LSDA_reactions-min.png)

**Fig. S5 LSDA reactions with different reaction volumes.** The reaction volume was varied to 5, 10 and 25 µL. Nt.BstNBI was used for all measurements. The LSDA reaction with 5 µL reaction volume could still give significant color change.

## Oxidation

### Confirmation of GQ-catalyzed TMB oxidation

*Text and figure adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08).*

Guanine-quadruplex (GQ) sequences were known to have peroxidase-like activity when associated with hemin3. This allowed GQ-hemin complexes to be used as a catalyst for peroxidation reactions as an alternative to horseradish peroxidases3,4. Due to their enzyme-like ability, GQ-hemin complexes are often referred to as GQ DNAzymes. Just like horseradish peroxidases, GQ-hemin complexes can catalyze the color-changing oxidation reaction of 3, 3’, 5, 5’-tetramethylbenzedine (TMB) in the presence of peroxide. However, different GQ sequences may harbor different enzymatic capabilities. Our preliminary analysis identified EAD2+3’A as the most potent GQ DNAzyme (Fig. 6). EAD2+3’A was thus selected as the final signal reporter.

![](https://static.igem.org/mediawiki/2020/1/1f/T--Leiden--Comparing_DNAzymes_over_time_650nm-min.png)

**Fig. 6 TMB oxidation rate of different GQ DNAzyme sequences.** Here, the catalytic activities of EAD2+3’A (BBa\_K3343000), BBa\_K1614007, and BBa\_K1614007+3’A (BBa\_K3343000) GQ DNAzymes in pH 6.0 phosphate buffer were compared. The DNAzyme sequences are shown in Table 1 on the [Parts](https://2020.igem.org/Team%3ALeiden/Parts) page. The concentration of DNAzyme was kept at 1 µM in all measurements. EAD2+3’A was identified as the most potent DNAzyme in catalysing TMB oxidation.

EAD2+3'A was added to the Registry as a New Part ([BBa\_K3343000](http://parts.igem.org/Part%3ABBa_K3343000)).
[BBa\_K3343001](http://parts.igem.org/Part%3ABBa_K3343001) was added to the Registry as an Improved Part .

![](https://static.igem.org/mediawiki/2020/a/ae/T--Leiden--Results12_Kinetic_parameters.png)

![](https://static.igem.org/mediawiki/2020/4/40/T--Leiden--Results13_TMB_Oxidation.png)

**Fig. 7 TMB oxidation by EAD2+3’A DNAzyme. Kinetic parameters of the DNAzyme were measured based on its ability to catalyze TMB oxidation reaction at different TMB concentrations.** In the figure, data points represent initial oxidation rate measured from each experimental replicate while the curve represents model-predicted kinetics at each given TMB concentration. Rate kinetics of EAD2+3’A were assumed to follow Michaelis-Menten kinetics. The initial rate of each individual replicates was calculated based on differences in oxidized TMB absorbance value at 650 nm for the first minute of the observation. The resulting model could sufficiently describe each individual replicate measurement (Fig. S6).

Characterization of EAD2+3’A catalytic activity was performed based on the DNAzyme’s ability to oxidize TMB in the presence of an excess amount of peroxide and different concentrations of TMB. All reactions were performed in a pH 6.0 phosphate buffer. The rate of TMB oxidation was then monitored based on oxidized TMB’s absorbance at 650 nm wavelength (molar absorption coefficient is 39,000 M-1 cm-1)5. Kinetic parameters of the DNAzyme were described using the Michaelis-Menten equation (Eq. 1), with *kcat* and *Km* representing the molar maximum of TMB conversion rate and TMB affinity of EAD2+3’A, respectively. The values of *kcat* and *Km* were calculated using multiple regression analysis by simultaneously minimizing the error of all measurement-simulation pairs. The approximated kinetic parameters of EAD2+3’A against TMB are shown in Fig. 7, Fig. S6, and Table 1. We used these parameters in our (semi-)empirical [model](https://2020.igem.org/Team%3ALeiden/Model) to simulate the underlying reaction kinetics of our detection technology.

![](https://static.igem.org/mediawiki/2020/a/ae/T--Leiden--Results12_Kinetic_parameters.png)

![](https://static.igem.org/mediawiki/2020/4/40/T--Leiden--Results13_TMB_Oxidation.png)

**Fig. 7 TMB oxidation by EAD2+3’A DNAzyme. Kinetic parameters of the DNAzyme were measured based on its ability to catalyze TMB oxidation reaction at different TMB concentrations.** In the figure, data points represent initial oxidation rate measured from each experimental replicate while the curve represents model-predicted kinetics at each given TMB concentration. Rate kinetics of EAD2+3’A were assumed to follow Michaelis-Menten kinetics. The initial rate of each individual replicates was calculated based on differences in oxidized TMB absorbance value at 650 nm for the first minute of the observation. The resulting model could sufficiently describe each individual replicate measurement (Fig. S6).

Supplementary figures (Fig. S6)
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![](https://static.igem.org/mediawiki/2020/1/1b/T--Leiden--Results14_TMB_Oxidation.png)

**Fig. S6 Predicted TMB oxidation kinetics by EAD2+3’A could sufficiently describe reaction rates of each individual reactions.** The values above each figures represented TMB concentration used in each observation. Observations obtained from each experimental replicates were represented as data points. Reaction rate between each two observed time points were assumed to be constant and resulted to a linear increase in A650 value. Lines shown in the figure represent the simulated increase in A650 value over the observation period at a given TMB concentration, as predicted by the fitted kinetic model (Fig. 7), and by using the average of each measurement replicates at time point zero as starting point of the simulation.

[Overview](#marker)
[Coupling the reactions](#marker)

## References

1. TwistDx™. TwistAmp DNA Amplification Kits Combined Instruction Manual. 2018. Retrieved from https://www.twistdx.co.uk/docs/default-source/RPA-assay-design/ta01cmanual-combined-manual\_revo\_v1-3b.pdf?sfvrsn=14
2. Crannell, Z. A., Rohrman, B. & Richards-Kortum, R. Equipment-free incubation of recombinase polymerase amplification reactions using body heat. PLoS One 9, e112146 (2014).
3. Li, W. et al. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: Adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity. Nucleic Acids Res. 44, 7373–7384 (2016).
4. Yang, X. et al. Characterization of G-quadruplex/hemin peroxidase: Substrate specificity and inactivation kinetics. Chem. - A Eur. J. 17, 14475–14484 (2011).
5. Liu, Y., Zhu, G., Yang, J., Yuan, A. & Shen, X. Peroxidase-like catalytic activity of Ag3PO4 nanocrystals prepared by a colloidal route. PLoS One 9, e109158 (2014).

Here we describe how the three main reactions of our detection method (RPA, LSDA and DNAzyme-catalyzed TMB oxidation) were coupled. The method’s feasibility was evaluated by passing the product of each preceding reactions to the next in a serial manner.

*Text and figures adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08).*

## Dithiothreitol (DTT) inhibits GQ oxidation but is inactivated at low pH

After coupling the reactions for the first time, a positive colorimetric signal could not be obtained using our original method and formulation (Fig. 8a and b). Interestingly, the signal could be obtained when RPA product was purified before being used in the subsequent LSDA reaction (Fig. S7). Thus, we suspected RPA mixture to contain a chemical(s) that may have inhibited LSDA and/or DNAzyme-catalyzed TMB oxidation.

The RPA mixture from TwistDx Basic Kit contained dithiothreitol (DTT), a strong reducing agent that is commonly mixed into protein formulations. The presence of DTT is known to improve protein stability by keeping the proteins’ monothiol groups in its reduced state1. Knowing its role as a reducing agent, we hypothesized that DTT carryover in TMB oxidation likely prevented the accumulation of oxidized TMB. Indeed, TMB oxidation with purified EAD2+3’A DNAzyme could not produce color change in the presence of DTT (Fig. S8). Therefore, removal or inactivation of DTT is essential for our DNA detection scheme to work.

The reducing power of DTT is known to be limited to more basic conditions2. Here, this characteristic of DTT was exploited as an alternative strategy to inactivate DTT during the DNAzyme-mediated TMB oxidation. Our original TMB oxidation formulation was performed using a pH 6.0 phosphate buffer. At this pH, we found that DTT hampers TMB oxidation (Fig. S8). Thus, we explored the possibility of DTT inactivation by performing DNAzyme-mediated TMB oxidation under different buffer conditions (Fig. 8c, d and e). Based on this comparison, phosphate-citrate buffer of pH 4.2 was identified to be the most suitable for TMB oxidation in the presence of DTT.

![](https://static.igem.org/mediawiki/2020/b/b7/T--Leiden--Results15_Effects_DTT_%28AandC%29.png)
![](https://static.igem.org/mediawiki/2020/0/06/T--Leiden--Results15_Effects_DTT-min.png)

**Fig. 8 Effect of DTT on TMB oxidation reaction. a and b)** RPA mixture component prevented color generation in TMB oxidation. **a)** TMB oxidation of LSDA-treated unpurified RPA products could not generate observable color change. The same was also observable from the negative controls (NC), which were performed in the absence of RPA target sequence. **b)** in a parallel measurement, color generation could be observed when RPA mixture was not present in the final TMB oxidation mixture. Background color generation from the blank control was also significantly higher than that observed from the test variables, suggesting the presence of a color generation-inhibiting component in the RPA mixture. **c, d and e)** Acidic pH eliminates DTT reducing power. Here, we investigated the suitability of acidic **c)** phosphate-citrate buffer and **d)** phosphate-citrate buffer for GQ DNAzyme-catalyzed TMB oxidation reactions by comparing its performances to **e)** the original pH 6.0 phosphate buffer. In general, lower pH seemed to be favourable for signal production.

![A](https://static.igem.org/mediawiki/2020/6/6f/T--Leiden--M_Results8a.png)
![B](https://static.igem.org/mediawiki/2020/1/1f/T--Leiden--M_Results8b.png)
![C](https://static.igem.org/mediawiki/2020/2/2a/T--Leiden--M_Results8c.png)
![D](https://static.igem.org/mediawiki/2020/e/e9/T--Leiden--M_Results8d.png)
![E](https://static.igem.org/mediawiki/2020/5/5e/T--Leiden--M_Results8e.png)

**Fig. 8 Effect of DTT on TMB oxidation reaction. a and b)** RPA mixture component prevented color generation in TMB oxidation. **a)** TMB oxidation of LSDA-treated unpurified RPA products could not generate observable color change. The same was also observable from the negative controls (NC), which were performed in the absence of RPA target sequence. **b)** in a parallel measurement, color generation could be observed when RPA mixture was not present in the final TMB oxidation mixture. Background color generation from the blank control was also significantly higher than that observed from the test variables, suggesting the presence of a color generation-inhibiting component in the RPA mixture. **c, d and e)** Acidic pH eliminates DTT reducing power. Here, we investigated the suitability of acidic **c)** phosphate-citrate buffer and **d)** phosphate-citrate buffer for GQ DNAzyme-catalyzed TMB oxidation reactions by comparing its performances to **e)** the original pH 6.0 phosphate buffer. In general, lower pH seemed to be favourable for signal production.

Supplementary figures (S7-8)
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![](https://static.igem.org/mediawiki/2020/4/47/T--Leiden--Result16_TMB_Oxidation.png)

**Fig. S7 TMB oxidation could be performed using LSDA product from purified RPA product.** TMB oxidation progressed at a significantly higher rate in the presence of LSDA product compared to when the reaction was spiked with pure 4 µM EAD2+3’A DNAzyme. This suggested LSDA’s effectivity in producing EAD2+3’A to more than 4 µM within the 40-minutes incubation time. Moreover, this suggested the importance of RPA mixture removal in order to obtain TMB oxidation signal.

![](https://static.igem.org/mediawiki/2020/4/4a/T--Leiden--Results17_DTT.png)

**Fig. S8 DTT inhibits TMB oxidation reaction by EAD2+3’A.** Absorbance signal at 650 nm of each spiked samples was not distinguishable from the background noise signal in the presence of 0.1 mM DTT.

## Coupling RPA, LSDA, and TMB oxidation

Our attempt to serially couple RPA, LSDA, and TMB oxidation reaction in a pH 4.2 phosphate-citrate buffer did not produce a significant color signal. Although the low-pH buffer reduced DTT activity, the oxidation reaction was still inhibited by the presence of unpurified RPA mixture. Fig. 9a and b showed how the increasing amount of RPA mixture reduced the color generation rate during the oxidation reaction. The pellet mixture was found to completely inhibit color generation when diluted by less than 200-fold.

To further attenuate the oxidation-inhibiting effect of the RPA mixture, we decided to reduce the amount of RPA and LSDA product used in each of its subsequent reactions in order to obtain a final RPA mixture dilution rate of at least 625-fold. Additionally, we decided to further increase the acidity of phosphate-citrate buffer used in the TMB oxidation reaction to a final pH of 3.8 to anticipate for potential pH increase when the solution was mixed with LSDA product, which had a slightly basic pH. Here we explored the effectivity of different dilution schemes from RPA to SDA and from SDA to TMB oxidation. Based on this analysis, a dilution of 10-fold during transfer from RPA to SDA and a subsequent dilution of 80-fold during transfer from SDA to TMB oxidation was found to be the most favourable dilution scheme (Fig. 9c, d, and e); the change in absorbance was similar to the positive control that did not contain any RPA or SDA reagents (no DTT).

Serial coupling of RPA, LSDA, and TMB oxidation was achieved by using the new dilution scheme and pH adjustment (Fig. 9c and d). The resulting color signal was significantly higher compared to the blank controls. With the new dilution method and low-pH buffer, the TMB oxidation-inhibiting effects of compounds in the RPA and/or SDA reactions were diminished. Subsequently, this allowed successful coupling of RPA, SDA and TMB oxidation in a serial manner.

![](https://static.igem.org/mediawiki/2020/b/bd/T--Leiden--Results18_Dilution-RPA-min.png)

**Fig. 9 Dilution of RPA mixture is required for signal generation in TMB oxidation reaction. a and b)** increasing the amounts of RPA mixture (not incubated, no template) was found to significantly reduce signal generation during the TMB oxidation reaction. In these reactions, 1 µM EAD2+3’A DNAzyme was used to catalyze the reaction. **c and d)** during TMB oxidation, a signal generation rate comparable to that achieved from 1 µM EAD2+3’A DNAzyme could be obtained by reducing the amount of unpurified RPA product that is passed on to the LSDA reaction to one-fourth of its amount in the original scheme. Within the observed RPA product concentration range, the initial TMB oxidation rate appeared to be correlated to the amount of unpurified RPA product used in LSDA. **e)** serial coupling of RPA, LSDA, and TMB oxidation using the updated dilution scheme and buffer could produce observable color change.

![A](https://static.igem.org/mediawiki/2020/e/e7/T--Leiden--M_Results9a.png)
![B](https://static.igem.org/mediawiki/2020/c/c1/T--Leiden--M_Results9b.png)
![C](https://static.igem.org/mediawiki/2020/2/2d/T--Leiden--M_Results9c.png)
![D](https://static.igem.org/mediawiki/2020/7/71/T--Leiden--M_Results9d.png)
![E](https://static.igem.org/mediawiki/2020/4/47/T--Leiden--M_Results9e.png)

**Fig. 9 Dilution of RPA mixture is required for signal generation in TMB oxidation reaction. a and b)** increasing the amounts of RPA mixture (not incubated, no template) was found to significantly reduce signal generation during the TMB oxidation reaction. In these reactions, 1 µM EAD2+3’A DNAzyme was used to catalyze the reaction. **c and d)** during TMB oxidation, a signal generation rate comparable to that achieved from 1 µM EAD2+3’A DNAzyme could be obtained by reducing the amount of unpurified RPA product that is passed on to the LSDA reaction to one-fourth of its amount in the original scheme. Within the observed RPA product concentration range, the initial TMB oxidation rate appeared to be correlated to the amount of unpurified RPA product used in LSDA. **e)** serial coupling of RPA, LSDA, and TMB oxidation using the updated dilution scheme and buffer could produce observable color change.

## DMSO reduced primer dimer formation during RPA and improved method accuracy

Our serial RPA-LSDA-TMB oxidation method could also generate color signal even when the RPA reaction was performed in the absence of the targeted sequence (Fig. 10a). The false-positive signal was not observable when RPA mixture was not incubated. Similarly, the signal could not be generated when (pure) RPA primers were used as templates for the LSDA reaction (Fig. S9). This pinpointed the origin for the false signal to an RPA product. We suspected RPA product(s) of primer complexes to somehow generate a nickase site and a GQ sequence at the correct order, thus allowing LSDA to generate GQ DNAzyme from this side-product.

Real-time RPA analysis using SYBR Green dye captured the amplification of an unknown product in the absence of target template (Fig. 10b). This confirmed that our RPA primers alone could generate an amplicon. Interestingly, we also found that the non-tailed forward primer, and not the tailed reverse primer, could also generate a background amplicon on its own, even though the product may differ from that produced in the presence of both the forward and reverse primers.

As an attempt to reduce non-specific amplicon generation by primer complexes, dimethyl sulfoxide (DMSO) was added to the RPA reactions. DMSO is a polar aprotic solvent. Its ability to dissolve both polar and non-polar compounds allows it to disrupt secondary structure formation of DNA strands. By weakening the hydrogen bonds between primers, the presence of DMSO may prevent primer (self-) annealing3,4. Here we exploit this capability of DMSO to weaken the non-specific primer-primer binding and complex formation (Fig. 10c). In this sense, DMSO acted much like a filter – eliminating background noise while allowing significant levels of true-positive signal to be further amplified. Here we identified 2.5% DMSO to be the most suitable for our RPA primer pair.

The inclusion of 2.5% DMSO in the RPA mixture was found to be effective in reducing the occurrence of false-positive signal from our sequence detection method. Significant attenuation of the true-positive signal was not observable, confirming the strategy’s effectivity to selectively filter out most false-positive amplicons (Fig. 10d). False-positive cases could nevertheless still be observable. We suspected this was due to the residual production of primer-associated amplicons. Although the current RPA condition could prevent false-positive signal production in RT-RPA, purified product from this reaction could still give a smear at approximately 100 bp region in agarose gel electrophoresis (Fig. S10). Nevertheless, the current method showed 33.3% false-positive and 33.3% false-negative rates based on production of absorbance signals higher than 0.15 (a.u.) within the given test period. This suggested the need for further improvements and technique optimization.

Interestingly, the maximum readout of true-positive signals was significantly lower compared to that observed in our previous experiments. This can be due to the interaction between DMSO and components of the LSDA and/or TMB oxidation reactions. Further analysis is required to pinpoint the cause of this signal attenuation and to further optimize our sequence detection technique.

![](https://static.igem.org/mediawiki/2020/b/b6/T--Leiden--Results19_DMSO-min.png)

**Fig. 10 DMSO reduced primer dimer formation during RPA and improved method accuracy. a and b)** RPA amplification product in the absence of target template can produce a positive signal. **a)** The average endpoint colorimetry readout of negative tests was approximately was approximately twice as high as was observed in the blank control. In this figure, error bars represented standard deviation of three (positive and negative tests) or two (blank and EAD2+3’A control) replicates. **b)** RT-RPA analysis confirmed the occurrence of background amplicon production in the absence of target template. Production of this background amplicon appeared to be dependent on the forward and not the reverse (tailed) primer. **c)** Addition of DMSO into RPA mixture could eliminate background amplicon generation. Though effective, high concentrations of DMSO (5% v/v or higher) may also attenuate amplicon generation in the absence of target templates. Here we identified 2.5% DMSO to be the most suitable to distinguish the presence of template during the RPA reaction. **d)** 2.5% DMSO could significantly reduce false-positive signal from the overall sequence detection method. Nevertheless, significant production of absorbance signal could still be observed 33.3% of the time. This may be due to residual production of false-positive-associated amplicons (Fig. S10). Average signals generated from positive- and negative-tests are shown for comparison with the EAD2+3’A and the blank control. Individual measurements are shown in Fig. S11.

![A](https://static.igem.org/mediawiki/2020/b/ba/T--Leiden--M_Results10a.png)
![B](https://static.igem.org/mediawiki/2020/b/b3/T--Leiden--M_Results10b.png)
![C](https://static.igem.org/mediawiki/2020/e/e2/T--Leiden--M_Results10c.png)
![D](https://static.igem.org/mediawiki/2020/8/81/T--Leiden--M_Results10d.png)

**Fig. 10 DMSO reduced primer dimer formation during RPA and improved method accuracy. a and b)** RPA amplification product in the absence of target template can produce a positive signal. **a)** The average endpoint colorimetry readout of negative tests was approximately was approximately twice as high as was observed in the blank control. In this figure, error bars represented standard deviation of three (positive and negative tests) or two (blank and EAD2+3’A control) replicates. **b)** RT-RPA analysis confirmed the occurrence of background amplicon production in the absence of target template. Production of this background amplicon appeared to be dependent on the forward and not the reverse (tailed) primer. **c)** Addition of DMSO into RPA mixture could eliminate background amplicon generation. Though effective, high concentrations of DMSO (5% v/v or higher) may also attenuate amplicon generation in the absence of target templates. Here we identified 2.5% DMSO to be the most suitable to distinguish the presence of template during the RPA reaction. **d)** 2.5% DMSO could significantly reduce false-positive signal from the overall sequence detection method. Nevertheless, significant production of absorbance signal could still be observed 33.3% of the time. This may be due to residual production of false-positive-associated amplicons (Fig. S10). Average signals generated from positive- and negative-tests are shown for comparison with the EAD2+3’A and the blank control. Individual measurements are shown in Fig. S11.

Supplementary figures (S9-11)
    +

---

![](https://static.igem.org/mediawiki/2020/4/4a/T--Leiden--20_False-positives.png)

**Fig. S9 False-positive signal originated from RPA side-product in the absence of target template DNA.** Without incubation, the same no-template RPA mix could not produce similarly high signal during TMB oxidation (triangles). The same is also true when either primer sets or RPA mixture was missing.

![](https://static.igem.org/mediawiki/2020/e/ed/T--Leiden--Results21_Faint_band.png)

**Fig. S10 Faint band at approximately 150 bp region could be observed from purified RPA product in the absence of target template and in the presence of 2.**5% DMSO. Although this band could only become visible at a higher resolution, this suggested residual production of false-positive-associated amplicons that may result to an increase in our method’s false-positive rate.

![](https://static.igem.org/mediawiki/2020/7/75/T--Leiden--Results22_2%2C5%25DMSO.png)

**Fig. S11 2.5% DMSO could significantly reduce false-positive signal from the overall sequence detection method. a)** Positive RPA reactions (with target sequence) and **b)** negative RPA reactions (without target sequence) were performed in presence of 2.5% DMSO and analyzed with LSDA and GQ-catalyzed oxidation. Average signals generated from each positive- and negative-tests are shown in Fig. 4d for comparison with the EAD2+3’A and the blank control.

[The  reactions](#marker)
[Discussion](#marker)

## References

1. Cleland, W. W. Dithiothreitol, a New Protective Reagent for SH Groups. Biochemistry 3, 480–2 (1964).
2. Lukesh, J. C., Palte, M. J. & Raines, R. T. A potent, versatile disulfide-reducing agent from aspartic acid. J. Am. Chem. Soc. 134, 4057–4059 (2012).
3. Hardjasa, A., Ling, M., Ma, K. & Yu, H. Investigating the Effects of DMSO on PCR Fidelity Using a Restriction Digest-Based Method. J. Exp. Microbiol. Immunol. 14, 161–164 (2010).
4. Nakano, S. ichi & Sugimoto, N. The structural stability and catalytic activity of DNA and RNA oligonucleotides in the presence of organic solvents. Biophysical Reviews 8, 11–23 (2016).

## Discussion

*Text adapted from [our preprint](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08).*

In this study, we established the basic design of a label-free method for the detection of targeted nucleotide sequence. The method utilized a primer-based isothermal DNA amplification reaction known as RPA to detect the presence of a targeted nucleotide sequence. However, the use of RPA as a point-of-care detection method is rather limited by its inability to produce visible signal on its own. Here we showed how RPA can be coupled to a colorimetric reaction catalysed by a DNA-based enzyme (DNAzyme) known as guanine-quadruplex (GQ). When associated with hemin, GQ DNAzyme can serve as a peroxidase. This allows it to be used as an alternative to horseradish peroxidase in common oxidation-based colorimetry reactions, such as the oxidation of TMB and ABTS.

The DNAzyme’s essence as a DNA allows it to be produced alongside target amplification in RPA reactions. Our sequence detection scheme, the production of GQ DNAzyme was incorporated by encoding the reverse complement of GQ DNAzyme to the 5’-end of the RPA reverse primers used in our RPA reactions. As a result, this strategy allows GQ DNAzyme to be produced only when amplicon is produced during the RPA reaction.

GQ DNAzyme performs its catalytic activity as a single-stranded DNA. This creates a need to dispatch one strand of the co-amplified dsDNA GQ sequence from the rest of the RPA amplicon. In our sequence detection scheme, this role was fulfilled by linear strand displacement amplification (LSDA) reaction. LSDA relies on the activity of a nicking endonuclease to generate a single-stranded cut at the 5’-end of the GQ sequence as well as strand displacement capability of Bst 2.0 polymerase. Although the nickase Nt.BstNBI was used in most of our experiments, LSDA may also be performed by using other nickases, such as Nt.BspQI1.

As a whole, our sequence detection method incorporates three steps of amplification, reporter generation, and signal readout/measurement. The three reactions were RPA, LSDA and GQ-catalysed TMB oxidation, consecutively. The three reactions were connected in a serial manner such that a proportion of the product of each preceding reaction was used in each subsequent reaction.

Our first attempt to integrate the three reactions revealed how the presence of a reducing agent (commonly included in enzyme formulations) may have inhibitory effect in our colour-producing oxidation reaction (Fig. 8a and b, Fig. S7). In our experiments, DTT, which was inevitably present in the RPA and LSDA reaction mixes, was identified as the main reducing agent that prevented colour formation during subsequent TMB oxidation reactions (Fig. S8). The presence of DTT thus demanded a strategy to remove or inactivate DTT for our three-step detection scheme to function as intended.

Past attempts to couple RPA with oxidation-based colorimetry may have depended on buffer exchange procedures to remove DTT from the subsequent oxidation reactions. Though highly effective, these methods required additional measures to immobilize the reporting peroxidase while the previous solvent is washed away2. We circumvented the need of this additional complexity by implementing a dilution scheme that limits the DTT concentration in the final oxidation reaction. Knowing DTT’s significantly lower activity at acidic pH, we further adjusted our oxidation buffer pH to 3.8. By implementing the dilution scheme and pH adjustment of the oxidation reaction, we successfully coupled the three reactions in a serial manner (Fig. 9c-e).

Despite successful integration of the three reactions, the technique presented significant false-positive signals in the absence of target sequence. Further investigation revealed the production of primer-associated amplicons as the source of this false-positive signal. Using RT-RPA analyses, we found how the addition of 2.5% (v/v) DMSO in RPA reaction to effectively reduce the occurrence of false-positive signals (Fig. 10c).

The oxidation reactions that followed RPA with 2.5% DMSO could not yield a sufficiently distinct true-positive signal when compared to the negative control and the blank controls. We hypothesize that this observation was the result of DMSO-GQ interaction. In this line, a study of Dhakal et al. (2013) reported that coexistence of parallel and non-parallel GQ species (1:1 ratio) was observed in the presence of crowded buffers with dehydrating cosolutes, such as 40% w/v DMSO3. The fact that complexes formed by hemin and antiparallel GQs have much lower peroxidase-mimicking activity than those formed by hemin and parallel GQs may explain the low readout signal that was observed when DMSO was included in the RPA reaction4. This revealed the need to further improve and optimize the presented sequence detection method to be used as a rapid diagnostic tool.

Despite the need for further optimization, the presented sequence detection method holds potential to be a rapid diagnostic tool. Its RPA-based detection allows it to be repurposed to target other DNA sequence by simply changing its primers, similar to the development of PCR-based tests. This allows rapid adaptation of the method to target newly emerging diseases. In comparison, development of antibody and/or antigen-based tests may require significantly more time and resources to be redeveloped to target a new disease5. Additionally, the reagent costs of the presented detection method are similar to commercial PCR-based tests (Supplementary data). However, unlike PCR-based tests, our method does not require extensive instrumentation to give a signal readout. Altogether, the method presents a potential to be a rapidly adaptable point-of-care diagnostic tool.

Future research may focus on combining RPA and LSDA into a one-pot reaction. The development of a one-pot reaction may further hasten the reactions’ incubation period and reduce the reagent costs (dNTPs, buffers, polymerase). Moreover, this would greatly simplify the hardware design when applied in point-of-care diagnostics. It may be necessary to replace the commercial TwistDx RPA kit with a custom-made RPA mix to optimize its composition for one-pot RPA and LSDA. With such custom-made mix, the concentration of DTT could be reduced to increase the amount of RPA product allowed in the oxidation reaction and increase the method’s sensitivity. Altogether, the presented sequence detection scheme presented a promising potency as rapid point-of-care diagnostic tool that may contribute in future attempts to diagnose, and by doing so, monitor and contain the spread of an infectious disease.

## Publication

If the pdf doesn't show, click [here](https://static.igem.org/mediawiki/2020/e/e0/T--Leiden--20201013_Rapidemic_Manuscript_FOR_THE_WIKI.pdf) to download the pdf of our preprint! Or read it at [BioRxiv](https://www.biorxiv.org/content/10.1101/2020.10.14.337808v1?s=08)

[Coupling the reactions](#marker)

## References

1. Joneja, A. & Huang, X. Linear nicking endonuclease-mediated strand-displacement DNA amplification. Anal. Biochem. 414, 58–69 (2011).
2. Jauset-Rubio, M. et al. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Sci. Rep. 6, (2016).
3. Dhakal, S. et al. Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Res. 41, 3915–3923 (2013).
4. Kong, D. M., Yang, W., Wu, J., Li, C. X. & Shen, H. X. Structure-function study of peroxidase-like G-quadruplex-hemin complexes. Analyst 135, 321–6 (2010).
5. Peeling, R. W., Murtagh, M. & Olliaro, P. L. Epidemic preparedness: why is there a need to accelerate the development of diagnostics? The Lancet Infectious Diseases 19, 172–178 (2019).

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We are the 2020 team of iGEM Leiden. With an interdisciplinary team of students we aim to develop a point-of-care rapid diagnostic tool for infectious diseases!

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