Team:CSMU Taiwan/Design

Our animated logo is keeping you company until the page has loaded.

Our animated logo is keeping you company until the page has loaded.

Design


Overview

Current UTUC detections, such as computer tomography, magnetic resonance imaging and ureteroscopy are not suitable for UTUC high-risk groups, therefore these diagnostic methods are not recommended to be applied for early detection or continuous tracking of UTUC. The defects should be solved, so CSMU_Taiwan attempted to solve those problems and bring a brand-new method to improve the UTUC detection, GotCha, based on Rolling Circle Amplification, can synthesize DNA as the report data. The readout of fluorescence indicates the density of microRNAs, which is proved as a biomarker to UTUC.



Picture
Picture
Picture


GotCha Design


  1. UTUC related microRNA Introduction
    2.

    MicroRNAs (miRNAs) are endogenous small non-coding RNAs that plays a crucial part in signal regulation in the body. In the field of cancer diagnosis recently, there has been large amounts of studies on detection methods branching from using miRNAs as biomarkers. [1] This is because the miRNA levels in the human body are strongly linked to cancer as miRNAs have been identified to act both as oncogenes and as tumour suppressors. Specifically in the field of UTUC detection, hsa-miR-664a-3p and hsa-miR-33b-3p have been shown to be expressed at elevated levels in blood serum compared to healthy individuals, with a 8.12 and 3.98 fold difference respectively. [2] The significant elevation in expression of these two miRNAs in human blood serum has made them great candidates for biomarkers in UTUC detection. However, as miRNA levels in human blood serum remain to be very minute, there needs to be amplification strategies employed to ensure accurate detection.



  2. RCA Introduction
    3.

    The concept of Rollling circle amplification (RCA) originated from Rolling Circle Replication in plasmids and bacteriophage DNA. It is a relatively new method of amplification in the field of synthetic biology. Despite this, it is widely promoted for its function due to the convenience it provides as compared to traditional amplification methods.
    Rolling circle amplification (RCA) is an isothermal enzymatic process where a short DNA or RNA primer is amplified to form a long single stranded DNA or RNA using a circular DNA template and special DNA or RNA polymerases. The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. The power, simplicity, and versatility of the DNA amplification technique have made it an attractive tool for biomedical research and nano-biotechnology. Traditionally, RCA has been used to develop sensitive diagnostic methods for a variety of targets including nucleic acids (DNA, RNA), small molecules, proteins, and cells. [3]

    For RCA utilisation, the following conditions must be met to establish a favourable environment for the reaction:

    1. Suitable type of DNA Polymerase and DNA Polymerase buffer
    2. Suitable sequence of DNA or RNA primer as a primer for start of DNA polymerase action in RCA
    3. Circular DNA template
    4. Deoxynucleotide Triphosphates (dNTPs)


    In RCA reactions, the most common DNA polymerases used for amplification purposes are namely Phi29, Bst, and Vent exo-DNA polymerase, while in the case of RNA amplification it is normally T7 RNA polymerase.


  3. Extraction Improvement: Magnetic Separation
    4.

    In more common ways of sequence amplification, there is a need for total DNA/RNA extraction for polymerase action to start. This is because there is a need to ensure that there are no other substances affecting polymerase action. However, the process of total RNA extraction is time-consuming and heightens risk of contamination. Therefore, we sought ways to avoid the need of total DNA/RNA extraction.
    Targetting hsa-miR-664a-3p and hsa-miR-33b-3p in blood serum samples, we found a way to avoid the need of total DNA/RNA extraction, not affecting polymerase action in the RCA mechanism. Leveraging the interaction and adherence between Strepavidin and Biotin, we propose a RCA method involving Strepavidin-coated Magnetic Beads that can avoid total DNA/RNA extraction. [4] To do so, we use an Immobilisation Probe (IMP) modified with biotin to anchor our circular DNA probe to the Streptavidin-coated Magnetic Bead, which is the template for phi29 polymerase to carry out RCA on. After phi29 polymerase has reached the IMP binding site, it will eliminate the IMP to continue with the elongation on the circular DNA probe. So far, combining traditional RCA methods with the usage of Streptavidin-coated Magnetic Beads can amplify specific miR signals in samples at room temperature. [5] The above is the core idea of our product we call "GotCha".

    After amplification of target miR signals by GotCha, we sought to find a way to quantify total DNA product from the RCA mechanism. Fluorescence testing is widely used in DNA and protein detection. In the context of our project of which DNA products are single-stranded, we selected Evagreen Dye as the fluorescent dye of our choice. [5] Using back-calculation of fluorescent absorbance value (see Proof of Conceptfor more information), we are able to derive the amount of hsa-miR-664a-3p and hsa-miR-33b-3p in blood serum samples.


Experiment Design


  1. GotCha formation binding site design
    2.

    For the functioning of the RCA mechanisms, we have chosen to use miR 664-3p and miR 33b-3p as the RNA primers to start RCA on our circular DNA probe template. [6] To do so, we have designed for our circular DNA probe to contain a sequence of bases complementary to our respective target miRNAs (For more information, please refer to Parts). On top of that, as mentioned above, we have also avoided incidence of more than two complementary bases to the target miRNA on parts of our circular DNA probe apart from miR binding site. The above two principles ensure accurate binding of target miRNA to the circular DNA probe.
    For more information on Circular DNA Probe Design, please refer to Model.


  2. Circular Probe Design
    3.

    In the design of our circular probe, we initially attempted to do so manually. However, through consultation with our graduated senior Chen Kuan-Lin (see Human Practices), we decided to design a software for Circular Probe Design curated to our project.
    In the Circular Probe Design software, to ensure GotCha will capture our selected miRNAs as expected and at the same time not detach easily, the Immobilisation Probe and miRNA have to bind accurately to binding sites on the circular DNA probe. Therefore, we designed our Circular DNA sequence such that there are no more than two complementary base pairs with Immobilisation probe and miRNA. This ensures high accuracy in binding of Immobilisation and microRNA to their respective binding sites.
    For more information on Circular DNA Probe Design, please refer to Model.


  3. Ligation Method
    4.

    For circularisation of our linear DNA, ligation of two ends of the linear DNA is required to synthesise our circular DNA probe. (For more information, please see Protocol) To achieve this, we looked up various ligases suitable for self-circularisation of linear DNA. Through our partnership with Mingdao High School (for more information please refer to Partnership), we narrowed our candidates down to T4 DNA ligase and Circligase. With much research and discussion with Mingdao, both teams chose T4 DNA ligase.
    T4 DNA ligase catalyzes the formation of a phosphodiester bond between 5' terminal phosphate and adjacent 3' hydroxyl termini in duplex DNA, RNA or DNA/RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA and some DNA/RNA hybrids. We chose it as it is the industry standard for performance and quality of ligation of DNA or RNA hybrids, and therefore extremely promising for DNA self-circularisation.
    For self-circularisation of linear DNA using T4 ligase, we utilise a T4 DNA ligation primer that is 26nt in length, complementary to 13 bases on the respective ends of the linear DNA. After which, the T4 Ligase will attach to the double stranded segment of the combination of linear DNA and ligation primer, and add a phosphodiester bond between the two ends of the the linear DNA, completing the self-circularisation of linear DNA into our circular DNA probe. [7]


  4. Hybridization & Immobolisation
    5.

    To anchor the circular DNA probe to the Streptavidin-coated magnetic bead, we designed an Immobilisation Probe (IMP), which is a linear sequence of ssDNA made up of an anchoring chain sequence and circular DNA binding site. The 3' end of the anchoring chain sequence is modified with biotin for adherance to the Streptavidin-coated magnetic bead, while the circular DNA binding site consists of 7 bases complementary to the IMP binding site on the circular DNA probe (for more information, please refer to Parts).
    To utilise these parts mentioned above to serve their function, firstly we need to hybridise the IMP and Circular DNA Probe through complementary base pairing at the circular DNA binding site on IMP. This step creates a hybrid of our linear IMP and circular DNA probe, forming an arm like structure whereby each arm can be used for miR capture.
    Following which, we need to immobilise the hybrid onto the Streptavidin-coated magnetic beads. This is done through the modification of 3' end of the IMP with biotin, which will allow the hybrid to adhere to the Streptavidin-coated magnetic bead. This step will then form our GotCha kit.
    After immobilisation of the hybrids onto magnetic beads, it is possible to do miRNA capture without total DNA/RNA extraction. This is because target miRNA capture is able to be anchored through magnetic attraction of the magnetic beads onto magnetic racks, allowing for convenient removal of other substances in the blood serum sample through washing.


  5. RCA protocol design
    6.

    For our RCA design, there were multiple factors taken into consideration.
    Firstly, we chose to use the phi29 polymerase for our RCA mechanism as it has a high rate of DNA elongation and duplication[9]. Additionally, as it has exonuclease activity, this allows for smooth running of RCA.
    In our reaction environment, phi29 polymerase will first detect the miRNA primer at the miRNA binding site on the circular DNA probe. Through elongating using free dNTPs in the environment, it will add bases onto the DNA product strand using complementary base pairing against the template strand, which is the circular DNA probe. After one round of replication is done, phi29 polymerase will remove the existing DNA product strand from the circular DNA probe using exonuclease activity, and a new round of replication will start. After a fixed amount of time, there will be a long strand of ssDNA as the DNA product[8].


  6. GotCha Efficiency examination (Variable)
    7.

    For the examination of GotCha efficiency, we initially designed our experiments based on the core RCA mechanism. However, as extra elements such as magnetic beads were introduced, we formulated the ideal protocol tailored to GotCha through our wet-lab experiments (see Protocol). However, due to time constraints and the pandemic, we were unable to completely test the efficiency of GotCha. Some potential problems with GotCha include: magnetic beads handling, detection region of miR levels, etc.
    We have designed experiments for efficiency testing (see Protocol), and hope that we will be able to complete it after the pandemic.


Detection Methods


  1. Dye Selection & microRNA to fluorescence relationship
    2.

    Regarding the selection of DNA dye used in our experiment, with the help of Mingdao High School (see Partnership), we chose Evagreen Dye as the fluorescent DNA dye of our choice to dye RCA ssDNA product.
    For the convenient usage of our product, we will establish a comparison table relating fluorescence absorbance to miR amounts present in blood serum.


  2. Serum application
    3.

    Lastly, we will carry out tests with human blood serum samples to test the accuracy and sensitivity of our kit.



References

  1. Peng, Y., Croce, C. The role of MicroRNAs in human cancer. Sig Transduct Target Ther 1, 15004 (2016).
  2. Tao, J., Yang, X., Li, P., Wei, J., Deng, X., Cheng, Y., Qin, C., Ju, X., Meng, X., Li, J., Gu, M., Lu, Q., & Yin, C. (2015). Identification of circulating microRNA signatures for upper tract urothelial carcinoma detection. Molecular medicine reports, 12(5), 6752–6760.
  3. Ali, M. M., Li, F., Zhang, Z., Zhang, K., Kang, D. K., Ankrum, J. A., Le, X. C., & Zhao, W. (2014). Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chemical Society reviews, 43(10), 3324–3341.
  4. Paul, A., Avci-Adali, M., Ziemer, G., & Wendel, H. P. (2009). Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX. Oligonucleotides, 19(3), 243–254.
  5. Mao, F., Leung, W. Y., & Xin, X. (2007). Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC biotechnology, 7, 76.
  6. Dundas, C. M., Demonte, D., & Park, S. (2013). Streptavidin-biotin technology: improvements and innovations in chemical and biological applications. Applied microbiology and biotechnology, 97(21), 9343–9353.
  7. Kuhn, H., & Frank-Kamenetskii, M. D. (2005). Template-independent ligation of single-stranded DNA by T4 DNA ligase. The FEBS journal, 272(23), 5991–6000.
  8. Johne, R., Müller, H., Rector, A., van Ranst, M., & Stevens, H. (2009). Rolling-circle amplification of viral DNA genomes using phi29 polymerase. Trends in microbiology, 17(5), 205–211.
  9. Mohsen, M. G., & Kool, E. T. (2016, November 15). The Discovery of Rolling Circle Amplification and Rolling Circle Transcription. Acc Chem Res. Retrieved February 1, 2021, from https://pubmed.ncbi.nlm.nih.gov/27797171/

About us

We are the 2021 iGEM Team of
CSMU Taiwan.

Follow us

Contact us

CSMU_Taiwan iGEM Team　
No. 110, Section 1, Jianguo North
Road, South District, Taichung City, 40201,Taiwan

Sponsor