Engineered Nucleic Acids as Tools to Develop Amplified Detection Platforms
Date Issued
2016
Date
2016
Author(s)
Jou, Amily Fang-Ju
Abstract
DNA nanotechnology, instead of exploring the genetic information that determine the biological meanings of every living organism, has been endowed with a revolutionary non-biological use that make DNA a versatile nanomaterial for engineering diverse static nanostructures and dynamic nanodevices. The aim of this work is to exploit the DNA nanotechnology for construction of novel amplified detection platforms based on nucleic acids. DNA alternation and aberrant expression of nucleic acids have been correlated with diseases and even cancers, such that tremendous efforts in finding reliable nucleic acid-based biomarkers as an index of a specific disease or cancer continue to devote. In spite of widespread application of PCR, the disadvantages, such as compromised sensitivity from contamination along with special apparatus for operating thermal cycling, motivate the development of alternative methods. The first work described an integrated detection system for identifying Alzheimer’s disease-related single nucleotide polymorphism (SNP). The system involved the utilization of toehold-mediated strand displacement reaction to differentiate SNP site, a nicking enzyme amplification reaction to amplify and translate the target input to an universal DNA translator, and a universal read-out electrochemical module to output a readable electro-response by a portable electro-analyzer. The results confirmed the feasibility of the platform for remarkable selective detection of target in discriminating the concurrence of target and interfering non-target. We substantiated the integrated system for the application in the determination of a single nucleotide change as an indicator of risk for Alzheimer''s disease. Recent studies have suggested the emerging role of microRNA in Alzheimer’s disease, and the roles of microRNA in regulating gene expression have also become promising disease-/cancer-associated biomarkers. Circulating microRNA in blood, in particular, prioritizes the construction of sensing systems in the recent trend. The second work focuses on the development of an amplified sensing platform for the detection of prostate cancer-related microRNA. A “turn-on” optical sensor of a quantum dot (QD) modified with a DNA sequence consisting of a probe sequence for the target microRNA, along with a protected signal-initiating primer for telomerase-assisted formation of DNAzyme-mimicking G-quadruplexes in the catalyzation of chemiluminescent output. The target recycling by duplex-specific enzyme (DSN) enabled the digestion of DNA probe, and followed by the exposure of primer for DNAzyme generation, led to a two-layer amplification for the sensitive detection of the miR-141. Excellent selectivity in discriminating the inter-, intra-family microRNA was verified. We also evaluated the clinical applicability of the optical genosensors in the detection of miR-141 in the sera sample of prostate cancer patients and healthy subjects, and the results were also compared with that using a commercial ELISA kit which is based on the detection of prostate-specific antigen (PSA). The optical genosensors demonstrated better differentiation ability relative to the gold standard (ELISA), showing potential as a reliable clinical diagnostics for prostate cancer screening. In the third work, we further explored the DNA nanotechnology to design a platform for cancer cell imaging and therapy by utilizing a cancer cell-specific surface nucleolin-triggered, cell surface-directed self-assembly of G-quadruplex by hybridization chain reaction. Specific targeted imaging based on DAPI staining was observed for MDA-MB-231 breast cancer cells rather than M10 normal epithelial cells. MDA-MB-231 breast cancer cells were found to be more susceptive to treatment of a model therapeutic agent for cancer cell, zinc protoporphyrin (ZnPP), as indicated by a significant decline in the number of viable cells. The co-treatment of ZnPP and the nucleolin-directed DNA remodeling resulted in a synergistic therapeutic outcome, confirming the importance of the surface remodeling as the result of enhanced therapeutic effect. This unique theranostic paradigm based on the engineering of the molecular hairpin probes holds great promise for simultaneous diagnosis of disease, and targeted drug delivery.
Subjects
Nucleic acids
Tools
Amplified
Detection
Platforms
SDGs
Type
thesis