Team:Leiden/Experiments
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Rapidemic
Experiments
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.
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 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 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.
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.
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, BBa_K1614007 and BBa_K3343001. (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.
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.
References
- Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA Detection Using Recombination Proteins. PLoS Biol. 4, e204 (2006).
- Zanoli, L. & Spoto, G. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices. Biosensors 3, 18–43 (2012).
- Higgins, M. et al. PrimedRPA: primer design for recombinase polymerase amplification assays. Bioinformatics 35, 682–684 (2019). https://doi.org/10.1093/bioinformatics/bty701
- Joneja, A. & Huang, X. Linear nicking endonuclease-mediated strand-displacement DNA amplification. Anal. Biochem. 414, 58–69 (2011).
- 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).
- 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).