Problem
We aim to establish a detection system for early-stage colorectal cancer as our goal for this year. Colorectal cancer ranks among the top three in both incidence and mortality rates in Taiwan, and it is also prevalent globally, ranking among the top five in terms of prevalence and mortality rates. Early detection of colorectal cancer significantly increases the chances of successful treatment. Therefore, if we can invent a tool or technique capable of detecting colorectal cancer at an early stage, it would effectively prevent the worsening of the condition and increase early treatment rates. [1]
Currently, the primary screening methods for colorectal cancer involve the use of fecal immunochemical test to detect stool. If blood is detected in the stool, further examination using colonoscopy is performed to observe the presence of polyps in the colon. However, there are several issues with the current screening methods. Firstly, the accuracy of fecal immunochemical tests in colorectal cancer first stage is 41.2%, which means there is a 58.8% misdiagnosis rate and insufficient precision. Secondly, colonoscopy, while it provides a direct view of lesions in the colon and enables biopsy, requires burdensome preparatory measures. Patients are required to consume a large amount of bowel preparation solution and undergo strict dietary restrictions and medication limitations. This process is often challenging, and the invasive nature of colonoscopy may decrease the willingness of the general public to undergo the examination. [2]
In summary, the current screening methods have drawbacks such as insufficient accuracy, burdensome preparation, and invasiveness. Therefore, we hope to establish a rapid, convenient, and accurate method for early detection of colorectal cancer.
Inspiration
When it comes to detection systems, the most crucial aspect is determining the target for detection. Therefore, in our iGEM case study, we continuously search for suitable biomarkers to serve as the detection target. In the iGEM-2022 Patras team [3], they utilized a novel biomarker, circular form RNA (circRNA), as the detection target for lung cancer. Subsequently, we conducted extensive research on circRNA and found that these circRNAs often exhibit abnormal expression levels in various diseases, making them a promising new biomarker as indicated by numerous literature sources [4]. However, there is currently limited research on detecting circRNA, and the detection methods can only conduct in a laboratory, making it challenging to generalize.
In recent years, due to the impact of the COVID-19 pandemic, we have become familiar with rapid screening assays. These assays can swiftly detect the presence of COVID-19 infection with just a few drops of bodily fluids. Inspired by this, we envisioned utilizing similar rapid screening methods to swiftly, conveniently, and accurately to detect circRNA within the human body.
Our Goal
We aim to establish a fast, convenient, and accurate detection system for early-stage colorectal cancer by measuring the expression levels of circRNA in patients' blood. This system enables the assessment of the risk of developing colorectal cancer.
Solution
Selection of circRNA
We have decided to research a blood-based cancer detection system that requires only a single tube of blood sample from patients to determine the presence of colorectal cancer. After conducting extensive data querying, we have identified circRNA in the blood as our primary target for investigation. Specifically, we will focus on two types of circRNA: hsa_circ_0101802 and hsa_circ_0004771, as potential biomarkers. These circRNAs have been observed to be upregulated in colorectal cancer patient's serum. Ranging between 0 and 1, a higher area under curve (AUC) value indicates higher sensitivity and specificity, better at identifying true positive and true negative samples. According to research, hsa_circ_0004771 hsa_circ_0101802 and have AUC value of 0.854 and 0.86, respectively. [5][6] Which demonstrated these two circRNAs have high specificity and sensitivity in clinical studies. This makes them highly suitable candidates for detecting the presence of colorectal cancer in patients.
Establishment of detection system assisted by synthetic biology
We employ synthetic biology techniques to engineer E. coli for the production of circRNA precursors (complementary DNA). Followed by circRNA synthesis, the resulting circRNA will serve as the detection targets in our testing system. The objective of this approach is to mimic the expression of circRNAs in patients' bodies and validate the effectiveness of our detection system
Detection system for circRNA
We have designed two methods for detecting the target cicRNAs: “Gold nanoparticle-based colorimetric assay” and “lateral flow rapid screening assay”.
Figure 1. Overall concept of our project. “RCA” is the abbreviation of “Rolling Circle Amplification”. “AuNPs” is the abbreviation of “Gold Nanoparticles”. “RT-RPA” is the abbreviation of “Reverse Transcription-Recombinase Polymerase Amplification”. “PCRD” is a kind of lateral flow strip.
1. Gold nanoparticle-based colorimetric assay
In the gold nanoparticle-based colorimetric assay, considering the circRNA may exist in low concentration in human serum, we incorporate an isothermal amplification method known as rolling circle amplification (RCA) to enhance the circRNA signal by producing long-repeated complementary DNA (cDNA) (Figure 2). (see page design for the mechanism of RCA)
Figure 2. A diagram shows how RCA amplify circular RNA by producing long repeated single-stranded complementary DNA.
Additionally, we introduce a single-stranded DNA oligo, referred to as a probe, to act as a bridge connecting the cDNA and gold nanoparticles. This design is effective because the probes are complementary to the RCA products.
The gold nanoparticle solution normally appears in red color. However, a color change to purple occurs when aggregations form upon the addition of salts [7]. Exploiting this characteristic, when a high level of circRNA is present in the tested sample and is amplified by RCA, the resulting cDNAs can specifically attach to the probe-conjugated gold nanoparticles. This inhibits the aggregation of gold nanoparticles, and the solution retains its red color when treated with salts. Conversely, if the tested sample contains a low level of expressed circRNA, the gold nanoparticle solution turns blue (Figure 3).
Figure 3. A diagram shows the mechanism of gold nanoparticle-based colorimetric assay.
2. Lateral flow test
In lateral flow test, we take advantage of a lateral flow strip, PCRD (Abingdon Health) [8]. PCRD includes one control (C) line and two test (T) lines. The purpose of these lines is to confirm the presence of circRNA in the sample and ensure the test's accuracy. For the function of PCRD, the target sequence should be modified with different tags both at the 3'- and 5'-ends. Initially, the sequences modified with biotin can bind to the anti-biotin on the surface of carbon particles in conjugated pad. Subsequently, sequences modified with DIG (or FAM) can bind to the anti-DIG (or anti-FAM) and become immobilized, resulting in the black color on the test line. For control line, the anti-biotin on the surface of carbon particles can attach to the anti-mouse, leading to the black color on the control line (Figure 4).
Figure 4. A diagram shows the mechanism of lateral flow strip (PCRD).
Reverse transcription-recombinase polymerase amplification (RT-RPA) is an isothermal amplification method that is introduced not only to amplify circRNA but also to incorporate modified primers to the amplicons. (see page design for the mechanism of RT-RPA). RT-RPA amplifies circRNA by producing double-stranded complementary DNA (cDNA); the resulting amplicons contain modified primers at the 3'- and 5'-ends, respectively (Figure 5).
Figure 5. The mechanism of recombinase polymerase amplification (RPA).
Followed by RT-RPA, the amplified product is dropped onto the purchased PCRD. If T line and C line both appear to be observed, indicating a high level of circRNA (indicating a risk of colorectal cancer) in the sample, while only C line observed represents a negative outcome (no disease).
Advantages and innovations
1. Our design, compared to existing methods for detecting colorectal cancer, requires only a single tube of blood sample for detection. This approach has a higher acceptance rate among the general public, and the use of two circRNAs ensures a high detection accuracy. Furthermore, our system allows for the early detection of the risk of colorectal cancer in patients.
2. Currently, there is a lack of literature on the rapid screening of circRNA. We have created two novel early colorectal cancer detection method, which make a significant contribution to future research in colorectal cancer-related diagnosis.
Click to go Design!Reference
[2] Chakrabarti, S., Peterson, C. Y., Sriram, D., & Mahipal, A. (2020). Early stage colon cancer: Current treatment standards, evolving paradigms, and future directions. World journal of gastrointestinal oncology, 12(8), 808-832. https://doi.org/10.4251/wjgo.v12.i8.808
[3] https://2022.igem.wiki/patras-medicine/model/
[4] Verduci, L., Tarcitano, E., Strano, S., Yarden, Y., & Blandino, G. (2021). CircRNAs: role in human diseases and potential use as biomarkers. Cell death & disease, 12(5), 468. https://doi.org/10.1038/s41419-021-03743-3
[5] Pan, B., Qin, J., Liu, X., He, B., Wang, X., Pan, Y., Sun, H., Xu, T., Xu, M., Chen, X., Xu, X., Zeng, K., Sun, L., & Wang, S. (2019). Identification of Serum Exosomal hsa-circ-0004771 as a Novel Diagnostic Biomarker of Colorectal Cancer. Frontiers in genetics, 10, 1096. https://doi.org/10.3389/fgene.2019.01096
[6] Bai, L., Gao, Z., Jiang, A., Ren, S., & Wang, B. (2021). circ_0101802 functions as a sponge of miR-1236-3p to facilitate the proliferation, migration and invasion of colorectal cancer via regulating MACC1. Journal of pharmacological sciences, 147(1), 104-113. https://doi.org/10.1016/j.jphs.2021.06.002
[7] Chang, C. C., Chen, C. P., Wu, T. H., Yang, C. H., Lin, C. W., & Chen, C. Y. (2019). Gold Nanoparticle-Based Colorimetric Strategies for Chemical and Biological Sensing Applications. Nanomaterials (Basel, Switzerland), 9(6), 861. https://doi.org/10.3390/nano9060861