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 page, on this page we outline the underlying thought process that led to our final engineering success.
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.
Cycle 1: The design of the mechanism
Research existing detection techniques
Text adapted from our preprint.
With the aim to develop a rapid, accurate, and accessible diagnostic kit, we narrowed our focus to point-of-care nucleic acid-based tests. 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?
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.
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.
(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.
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.
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.
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.
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.
Fig. 6 DTT reduces protein thiol groups. Figure adapted from https://commons.wikimedia.org.
Imagine and design strategies to eliminate inhibition by DTT
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.
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.
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.
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), 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.
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).
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.
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.
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!
- Yan, L. et al. Isothermal amplified detection of DNA and RNA. Molecular BioSystems 10, 970–1003 (2014).
- Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000).
- 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).
- 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).
- Walker, G. T. et al. Strand displacement amplification - an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20, 1691–1696 (1992).
- 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).
- Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, e204 (2006).
- Zanoli, L. M. & Spoto, G. Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors 3, 18–43 (2013).
- 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).
- Kozwich, D. et al. Development of a novel, rapid integrated Cryptosporidium parvum detection assay. Appl. Environ. Microbiol. 66, 2711–2717 (2000).
- 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).
- Peng, H. et al. DNAzyme-Mediated Assays for Amplified Detection of Nucleic Acids and Proteins. Analytical Chemistry 90, 190–207 (2018).
- Roembke, B. T., Nakayama, S. & Sintim, H. O. Nucleic acid detection using G-quadruplex amplification methodologies. Methods 64, 185–198 (2013).
- 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).
- 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).
- 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).
- 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).
- Nie, J. et al. A G-quadruplex based platform for label-free monitoring of DNA reaction kinetics. Analyst 143, 1444–1453 (2014).
- 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).
- 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).
- 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).
- Cleland, W. W. Dithiothreitol, a New Protective Reagent for SH Groups. Biochemistry 3, 480–2 (1964).
- 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).
- Chakrabarti, R. & Schutt, C. E. The enhancement of PCR amplification by low molecular-weight sulfones. Gene 274, 293–8 (2001).
- 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).
- 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).