Inspiration

Early diagnosis of cancer

Cancer, a devastating health issue affecting 14.1 million new cancer cases worldwide in 2012, is one of the killers to human beings.1 Early diagnosis is recommended to effectively reduce the mortality rate and life expectancy of patients. For instance, in the U. K., nearly 90% of patients diagnosed with stage I lung cancer lived for more than a year while only 19% of patients diagnosed at stage IV do so.2 Early diagnosis for cancer is also believed to be vital for successful treatment and recovery.

Significant gene mutations inside body might signal the potential of development of cancers. Although there are current research that diagnoses cancer(s) by analyzing individual genetic mutation profiles3,4, but such diagnostic methods take up considerable durations of time for accurate results. While conventional diagnostic methods involve complicated procedures, a simple, rapid and specific DNA nanostructure probe is introduced for the detection of cancer biomarkers to facilitate diagnosis. Research background - DNA nanostructures and miRNAs as biomarkers DNA has emerged as a promising material that allows researchers to construct novel designs as its structure could be easily and accurately predicted.5 Examples of DNA nanostructures include nano-tweezers to detect norovirus and a DNA ‘Nano-Claw’ to detect membrane markers of cancer cells.6,7

DNA boolean logic gates are constructed to generate outputs depends on the presence of multiple targets with a variety of probes, including OR-gates and AND-gates which generate fluorescence resonance energy transfer (FRET) signal output in regard to the presence of inputs through hybridization of target with the probe.8

Among the targets, a range of miRNAs have been identified to be associated with cancer. To illustrate, miR-15b-5p, miR-338-5p, and miR-764 found in plasma are potential biomarkers for detecting hepatocellular carcinoma cancer (HCC), a common type of liver cancer.9 able to detect promising biomarkers - micro RNAs (miRNAs)15 to provide meaningful results.

Objectives

in vivo synthesis of functional DNA nanostructure

We design a novel DNA nanostructure that detects multiple miRNA targets at the same time, thus avoid false positives by promoting discrimination of our detection system. The nanostructure could specify a single base mutation of miRNA while maintaining a relatively low threshold. The logic is simple - A tool which detect a combination of biomarkers eliminates the possibility of other cancer diseases.

In regard to recent in vitro applications of DNA nanostructure to achieve point-of-care (POC) diagnostics17, we develop a self-assembling DNA nanostructure potentially to target a mRNA in vivo. With the capacity of synthesizing and assembling within a cell, the need of target amplification is omitted based on the richer mRNA content in the cell than in serum by folds. In contrast, molecular beacons with its ends linked to a fluorophore and a quencher18, however, do not hold the capacity to be synthesized within a cell. Without attaching to a fluorophore and a quencher mean a significantly lower cost. [[File:HKU_ProjectDescription_Mindmap1.jpg]

Current progress

Our design is a self-assembling oligonucleotides as the functional unit. Our aim is to construct a nanostructure can detect at least two miRNA biomarkers such that our design can reach a higher accuracy for diagnosis. For the selection of biomarkers, we are looking for a combination of miRNAs that is specific to diseases.

At current stage, we test different designs in vitro first to see if they can produce desired signals. After proving our designs can work in vitro, we will attempt to test them in vivo. Finally, we will try to design a mechanism such that E. coli can synthesize these oligonucleotides required to form the specified nanostructure.

Signifcances

A leap forward - in vivo synthesis of 3D functional DNA nanostructures

In the past decade, functional DNA nanostructures have been used in similar in vitro approaches to detect various cancer biomarkers.10,11 It is noted that most of those designs were applied in vitro. Recently, 1D and 2D DNA structures were successfully expressed and assembled in vivo,12 while several novel 3D DNA structures were synthesized to produce signals in vivo.13,14 So given these advancements, our ultimate goal is to allow our functional DNA nanostructure to be synthesized and self-assembled in E. coli, that can function inside the disease cells. This, if successful and with further refinement, could be a great replacement to colour coded surgery in the surgical field.16

Last but not least, the cost and quality of production, efficiency and accuracy of our intracellularly-synthesized 3D structure will be compared to current diagnostic methods.

References

References American Cancer Society. (2015). Global Cancer Facts & Figures. Retrieved from http://www.cancer.org/acs/groups/content/@research/documents/document/acspc-044738.pdf Public Health England. (2014). National Cancer Intelligence Network Cancer survival in England by stage. Retrieved from http://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/lung-cancer/survival#ref-3 Pereira, B., Chin, S., Rueda, O. M., Vollan, H. M., Provenzano, E., Bardwell, H. A., Pugh, M., et al. (2016). The somatic mutation profiles of 2500 primary breast cancers refine their genomic landscapes. Nature Communications Pereira, B., Chin, S. F., Rueda, O. M., Vollan, H. K. M., Provenzano, E., Bardwell, H. A., ... & Tsui, D. W. (2016). The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes. Nature communications, 7. 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. 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. 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. 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. 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. Miao, P., Wang, B., Chen, X., Li, X., & Tang, Y. (2015). Tetrahedral DNA nanostructure-based microRNA biosensor coupled with catalytic recycling of the analyte. ACS applied materials & interfaces, 7(11), 6238-6243. Li W. et. al. (2015). Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate. Biosensors and Bioelectronics. 71, 401-406. Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7. Kim K. et. al. (2013). Drug delivery by self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells. Chem. Commun. 49, 2010-2012. Kim K. et. al. (2013). Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron. Biomaterials. 34, 5226-5235. Montani, F., & Bianchi, F. (2016). Circulating Cancer Biomarkers: The Macro-revolution of the Micro-RNA. EBioMedicine, 5, 4-6. Nguyen, Q. T., & Tsien, R. Y. (2013). Fluorescence-guided surgery with live molecular navigation [mdash] a new cutting edge. Nature reviews cancer, 13(9), 653-662. Hartman, Mark R., et al. "Point-of-care nucleic acid detection using nanotechnology." Nanoscale 5.21 (2013): 10141-10154. TSOURKAS, Andrew, et al. Hybridization kinetics and thermodynamics of molecular beacons. Nucleic acids research, 2003, 31.4: 1319-1330.