Team:HK HCY LFC/Design

Team:HK HCY LFC - 20118.igem.org

Design

Self-assembled DNA Tweezer Nanomachine

In a biosensor, a bioreceptor interacts with the analyte and this interaction will be converted into a measurable signal by a transducer.

In our design, a split DNA aptamer is integrated into G-quadruplex tweezers to detect the miRNA 25. It is found that comparing with various DNA machine mechanisms, DNA tweezers hold some specific advantages for sensing applications as the mechanism does not involve strand displacement and the structure is formed from only a small number of single-stranded DNA molecules. [1] Besides, it is believed that aptamer-based sensors act as a better sensor comparing to antibodies because they are less expensive, more stable than antibodies. Similarly, comparing with antibody-antigen interaction, aptamers also show high thermostability and specificity in binding the miRNA 25 through Watson- Crick base pairing. [2, 3]

Regarding to reporting system that enables exhibition of results, G-quadruplex, which mediated peroxidase activity with hemin can be observed colorimetrically, could be adopted into the tweezer well. Therefore, a quadruplex-hemin reporting system is ideal compared to fluorescence as it is relatively low-cost and visualizable by eye for potential future diagnostic application in a rapid diagnostic device. [4, 5]

Tanner and co-workers demonstrated that a DNA tweezers nanomachine can successfully recognize the Plasmodium falciparum lactate dehydrogenase. In their design, a split DNA aptamer is integrated into a G-quadruplex tweezers and closing of the tweezers enables G-quadruplex hemin mediated peroxidase activity. [5] A similar nanostructure is designed in our project for the recognition of has-mir 25.

Tiamat, which is a tool to design large and complex DNA nanostructure, is used to design our nanostructure [6]. According to figure 1, the nanostructure designed consists of three single strand DNA oligos. DNA oligo 1 refers to the longest one in the middle; DNA oligo 2 refers to the one on the left while DNA oligo 3 refers to the one on the right. It is showed in figure 2 that DNA oligo 1 consists of a split G-quadruplex which is labelled green at both ends; DNA oligo 2 and 3 consists of split aptamer which are labelled blue and red respectively. The sequences of each DNA oligo are shown in figure 3. The three strands assembled through complementary base pairing in which adenine (A) pairs with thymine(T) while guanine(G) pairs with cytosine(C).

Figure 1: Drawing of designed DNA nanostructure using Tiamat

Figure 2: Designed DNA nanostructure with sequences of each strand shown. The complementary regions to part of the has-mir 25 are located at the 3’ end of DNA oligo 2 and 5’ end of DNA oligo 3 respectively (labelled blue and red ). The split G-quadruplex is located at both ends of DNA oligo 1 (labelled green)

Figure 3: The mechanism of DNA nanostructure generating detectable signals (G-quadruplex) upon binding with the target RNA oligo (has-mir 25)

References
[1] Angell, C., Kai, M., Xie, S., Dong, X., & Chen, Y. (2018). Bioderived DNA Nanomachines for Potential Uses in Biosensing, Diagnostics, and Therapeutic Applications. Advanced healthcare materials, 7(8), 1701189.
[2] Kilic, T., Erdem, A., Ozsoz, M., & Carrara, S. (2018). MicroRNA biosensors: Opportunities and challenges among conventional and commercially available techniques. Biosensors and Bioelectronics, 99, 525-546.
[3] Azimzadeh, M., Rahaie, M., Nasirizadeh, N., Daneshpour, M., & Naderi-Manesh, H. (2017). Electrochemical miRNA biosensors: the benefits of nanotechnology. Nanomedicine Research Journal, 2(1), 36-48.
[4] Wu, Y., Zou, L., Lei, S., Yu, Q., & Ye, B. (2017). Highly sensitive electrochemical thrombin aptasensor based on peptide-enhanced electrocatalysis of hemin/G-quadruplex and nanocomposite as nanocarrier. Biosensors and Bioelectronics, 97, 317-324.
[5] Shiu, S. C. C., Cheung, Y. W., Dirkzwager, R. M., Liang, S., Kinghorn, A. B., Fraser, L. A., ... & Tanner, J. A. (2017). Aptamer‐Mediated Protein Molecular Recognition Driving a DNA Tweezer Nanomachine. Advanced Biosystems, 1(1-2), 1600006.
[6] Williams, S., Lund, K., Lin, C., Wonka, P., Lindsay, S., & Yan, H. (2008, June). Tiamat: a three- dimensional editing tool for complex DNA structures. In International Workshop on DNA-Based Computers (pp. 90-101). Springer, Berlin, Heidelberg.