Team:Hong Kong HKU/projectoverview

Planning and Design

After extensive brainstorming and research, our DNA nanostructure was designed with the help of the software, Tiamat.

Synthesis of DNA Nanostructure

Our two-dimensional DNA nanostructure was synthesised through thermal annealing of the six individual oligonucleotide strands. The hydrogen bonds of the DNA melt at a high temperature in the thermal cycler and upon cooling, the most stable structure, which is our desired nanostructure, is formed.

Observation of DNA Structure Formation

Polyacrylamide gel electrophoresis (PAGE) was carried out to assess the complementary binding of the structure oligonucleotides and the successful formation of the structure. Interaction and binding of oligonucleotides cause gel bands to move up due to higher molecular size.

Detection of Target

Fluorometric assay was carried out to evaluate the effectiveness of the DNA nanostructure in detecting the Huntington’s disease miRNA biomarker, Hsa-miR-34b. The two-dimensional DNA nanostructure changes to a three-dimensional structure on detection of target.

Evaluation and Improvement

Input was received from a number of potential end-users of a diagnostic test made using our DNA nanostructure, for instance through questionnaires sent to medical professionals. All feedback was taken into account and incorporated into the redesigning and improvement of our structure.

Synthesis of Biobricks

Finally, five Biobricks were produced, which can produce the five single-stranded DNA oligonucleotides of our structure respectively. The ssDNA can come together to form our functional tetrahedral structure in the presence of the specific target.

References:

1. Fo, W. (2007). Huntington’s Disease. The Lancet, 369(9557), 218-228
2. https://www.qiagen.com/us/shop/pcr/primer-sets/rt2-profiler-pcr-arrays/?catno=PAHS-123Z#resources.
3. Thiviyanathan, Varatharasa, and David G. Gorenstein. "Aptamers and the next generation of diagnostic reagents." PROTEOMICS-Clinical Applications 6.11-12 (2012): 563-573.
4. Chen, Y. J., Groves, B., Muscat, R. A., & Seelig, G. (2015). DNA nanotechnology from the test tube to the cell. Nature nanotechnology, 10(9), 748-760.
5. Nakatsuka, K., Shigeto, H., Kuroda, A., & Funabashi, H. (2015). A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids. Biosensors and Bioelectronics, 74, 222-226.
6. You, M., Peng, L., Shao, N., Zhang, L., Qiu, L., Cui, C., & Tan, W. (2014). DNA “nano-claw”: logic-based autonomous cancer targeting and therapy. Journal of the American Chemical Society, 136(4), 1256-1259.
7. Pei, H., Liang, L., Yao, G., Li, J., Huang, Q., & Fan, C. (2012). Reconfigurable Three‐Dimensional DNA Nanostructures for the Construction of Intracellular Logic Sensors. Angewandte Chemie, 124(36), 9154-9158.
8. Chen, Y., Chen, J., Liu, Y., Li, S., & Huang, P. (2015). Plasma miR-15b-5p, miR-338-5p, and miR-764 as Biomarkers for Hepatocellular Carcinoma. Medical science monitor: international medical journal of experimental and clinical research, 21, 1864.
9. Montani, F., & Bianchi, F. (2016). Circulating Cancer Biomarkers: The Macro-revolution of the Micro-RNA. EBioMedicine, 5, 4-6.
10. Kantcheva, R.B. (2013). Identification and analysis of genetic modifiers of mutant huntingtin toxicity in Saccharomyces cerevisiae. Diss. University of Leicester.