Contribution
This year, our team’s contribution is based on the 2020 Worldshaper-Shanghai team, 2024 cjuh-jlu20-china team, and WageningenUR team, which all used technology for disease detection based on RNA secondary structure.
Toehold switches are a class of riboregulators that can be activated when interacting with a specific trigger RNA [1]. Each toehold switch contains a stable hairpin loop that includes a ribosome binding site (RBS) and a start codon among other base-pairing interactions. Downstream of the hairpin loop is the coding sequence of a gene. The stable loop structure prevents the binding of ribosome, and as a consequence, translation of the downstream gene is repressed. The upstream region of the hairpin loop contains a single-stranded toehold domain that is designed to be complementary to part of the trigger RNA to be detected, with the remaining part of the trigger RNA complementary to the domain right next to the toehold. When the trigger RNA is present and interacts with the toehold domain, the hairpin loop is unfolded, exposing the RBS and the start codon, which permits translation of the downstream gene (Figure 1).
Figure 1. Sequence components and secondary structure of a toehold switch
The Loop-Initiated Isothermal RNA Activator (LIRA) is a novel RNA detection technology based on RNA secondary structure[2]. The single-arm structure of LIRA consists of a loop and complementary paired stem, with its recognition domain divided into two parts located within the stem (b*) and the loop (a*). LIRA hides the ribosome binding site (RBS) sequence and the start codon AUG necessary for RNA translation within the stem-loop structure. When the target miRNA is present, it binds to the LIRA recognition domain, disrupting its original structure and exposing the RBS and AUG, thereby initiating the translation of downstream reporter genes (Figure 2).
Figure 2. Sequence components and secondary structure of LIRA.
Many researchers have successfully applied these methods to disease detection, such as lung, kidney disease. Most importantly, they have revised the methods to achieve more precise diagnosis, which has inspired our work on improving breast cancer diagnosis. We summarized the research of previous teams on disease detection based on RNA secondary structure. The parts and their respective functions are shown in Table 1.
Table 1. Parts and their functions.
|
Part name |
Target |
Method |
Disease |
Color |
|
BBa_K3577000 |
PCA3 /KLK3 |
Toehold switch |
prostate cancer |
green |
|
BBa_K5038020 |
miR-142-3p /miR-210-3p |
LIRA |
myocardial infarction |
green |
|
BBa_K5106007 |
hsa-miR-484 |
Toehold switch |
Multiple Sclerosis |
Purple |
The table outlines specific genetic constructs, referred to as parts, that are engineered to target particular biological pathways involved in diseases. For example, BBa_K3577000 is designed to interact with PCA3/KLK3, biomarkers associated with prostate cancer. This construct employs a toehold switch mechanism, which allows for the precise regulation of gene expression in response to specific RNA signals, making it a valuable tool for detecting cancerous conditions. Another part, BBa_K5038020, targets the miR-142-3p and miR-210-3p microRNAs, which play critical roles in cellular responses to myocardial infarction, facilitating the understanding of heart tissue repair mechanisms. Lastly, BBa_K5106007 targets hsa-miR-484, implicated in multiple sclerosis, also utilizing a toehold switch to modulate gene expression.
In 2020, Worldshaper-Shanghai team applied toehold switch technology to diagnose early prostate cancer, which has been validated as non-invasive and sensitive. They used PCA3 (Prostate Cancer Antigen 3) and KLK3 (Kallikrein-Related Peptidase 3) as biomarkers, and the toehold switch (Part: BBa_K3577000) provides a visible readout if the user has prostate cancer. Not only did the patients’ samples containing PCA3 products and toehold plasmids show qualitative brilliant fluorescence (Figure 3). Additionally, they created a standard curve to quantify PCA3 products (Figure 4).
Figure 3. The verification of in vitro transcription/translation involving PCA3 products and toehold plasmids.
Figure 4. The parametric regression model between PCA3 copies and fluorescence values.
The curve appears to follow a sigmoid shape, indicating that initially, there is a gradual increase in fluorescence with low PCA3 copy numbers. As the amount of PCA3 mRNA copies increases, the fluorescence values rise as well, indicating a positive correlation. This trend suggests that higher levels of PCA3 mRNA led to increased fluorescence, likely due to enhanced binding activity or expression levels detected by the fluorescence measurement.
In 2024, cjuh-jlu20-china team applied a Loop-Initiated RNA Activator (LIRA)-based AND logic gate system to warn myocardial infarction patients of their risk of cancer[3]. The system can detect the biomarker microRNA (miRNA) and integrates with a cell-free system (Part: BBa_K5038020).
Figure 5. Different stem-loop ratio LIRA data
This used miR-142-3p and miR-210-3p as targets, which are highly expressed in post-MI patients. Different stem-loop ratios could influence the detection efficiency (Figure. 5).
Figure 6. Double-arm LIRA Sequences
Double-arm LIRA (Figure 6), which could detect miR-210-3p and miR-142-3p simultaneously, improves detection accuracy. They creatively combined the double-arm LIRA and the simplicity of a cell-free system to screen high-risk tumor individuals among patients after myocardial infarction, which will be extremely beneficial for patients in clinical settings.
In 2024, the WageningenUR team applied miRNAs as biomarkers for Multiple Sclerosis (MS) diagnosis[4]. They detected miRNA using toehold switches and logic gates (Part: BBa_K5106007). They designed the toeholds specific to hsa-miR-484. This miRNA was shown to be upregulated in the blood of multiple sclerosis patients. Three toeholds were designed to detect this miRNA, namely Toehold A, Toehold B, and Toehold C. According to their results, the toehold switch secondary structure can influence the miRNA combination with the miRNA (Figure. 7-8). Moreover, logic gates AND indicate two input triggers. This means that both triggers need to be present before an output signal could be observed. Most importantly, to ensure that the test meets our accessible requirements, they developed a paper-based blood test. They add the components necessary for establishing the threshold and the detection of the trigger miRNAs (Figure 9).
Figure 7. Cell-free expression (PURExpress) of toehold switch constructs in 2 μL reaction volumes
The picture shows a setup for detecting specific miRNAs using toehold switch constructs in a cell-free expression system, aimed at diagnosing Multiple Sclerosis (MS). The samples include controls and three toeholds (A, B, C) designed to detect hsa-miR-484, which is up-regulated in MS patients. The toehold switches only produce a detectable signal when they bind to their target miRNA, demonstrating specificity. The presence of logic gates, particularly the AND gate, ensures that an output signal is generated only when both input triggers are present. This setup emphasizes the potential for developing a paper-based blood test to accurately identify MS through targeted miRNA detection.
Figure 8. Absorption of Chlorophenol Red at 570 nm over time
The graph illustrates the absorption of Chlorophenol Red at 570 nm over time for different toehold switch constructs (TH A, TH B, and TH C) with and without the corresponding triggers. The solid lines represent the conditions where the triggers are present, while the dashed lines indicate the absence of triggers.
Figure 9. Cell-free expression (PURExpress) of toehold switch constructs in 2 μL reaction volumes.
The image depicts the cell-free expression of toehold switch constructs, highlighting Toehold Switch B and an AND gate configuration. The visual resembles a traffic light, with multiple purple markers representing the presence of different triggers. In this context, the AND gate indicates that both input triggers must be present for the output signal (indicated by the purple color) to be activated.
Future aspects
This year, our YiYe-China team is utilizing the toehold switch to diagnose breast cancer. Through our contribution, the following factors need to be taken into consideration when we want to improve the detection efficiency. We hope to make further efforts in the following areas to make the project more complete:This year, our YiYe-China team is employing the toehold switch for breast cancer diagnosis. In order to enhance detection efficiency, the following factors need to be considered based on our work. We aim to make further efforts in the following aspects to improve the completeness of the project:
Toehold switch structure - The software can successfully predict toehold switch sequences that fully anneal to the miRNA, with the hybridization reaction being energetically favorable. Different toehold switch structures showed different binding affinities to the same miRNA.
And gate – A two-input AND gate is a single toehold switch activated by two trigger RNAs. The input for the corresponding toehold switch consists of two input RNA molecules that are partially complementary to each other.
Stem-loop ratios –Regarding the trigger RNA sequence, the ratio of nucleotides in the recognition region that form part of the stem structure of LIRA to those in the recognition region that form part of the loop structure (stem-loop ratio) is critical for LIRA function. This ratio determines the efficiency of LIRA's interaction with its target RNA.
Cell Free system - Cell-free reactions are assembled by mixing the two PURExpress solutions (which contain cell-free reaction components) with the substrate, DNA template, and RNase inhibitor. These reactions are then applied to the discs.
Reference
1. Andrew Ching-Yuet, T., David Ho-Ting, Chu,Angela Ruoning, Wang et al., A comprehensive web tool for toehold switch design. Bioinformatics, 2018. 34.
2. https://2024.igem.wiki/wageningenur/eng.
