Novel DNA Self-assembly Modules:Construction, Hybridization Properties And Analytical Applications

The superb biocompatibility, chemical stability, facile modification and sequence programmability of DNA oligonucleotides have turned them into an ideal material for the explorations of various applications in biosensors and nanofabrications, This thesis is focused on the development of novel DNA-based self-assembly modules, and the investigation of their hybridization kinetics and analytical applications, which are detailed as follows:Nanoparticles conjugated with a single or multiple DNA oligonucleotides have found various applications in self-assembly based material fabrication, drug delivery, chemo-and bio-sensing, and bio-imaging. Due to the unique plasmonic property, enhanced Raman scattering, antibacterial activity, and catalytic and luminescent properties, silver nanoparticles have received great attention in nanomaterials science. However, the weak chemical and colloidal stability of silver nanoparticles are two major obstacles during their conjugation with DNA. Therefore, developing a low cost and efficient synthetic strategy for stable silver nanoparticles and exploring their valence-controlled DNA decorations will be a significant but challenging work. In this thesis, we used mechanically shortened fish sperm DNA (FSDNA) for the synthesis of ultra-stable water-soluble silver nanoparticles. Due to the good nucleation and size control of FSDNA, the as-synthesized silver nanoparticles had a mean diameter of2nm with a relatively narrow size distribution. As FSDNA could provide an excellent stabilization effect for nanoparticles, the synthesized silver nanoparticles were highly resistant to strongly ionic solutions containing up to3M Na" or300mM Mg2+, which was of great significance for DNA-based nanoparticle assembly since a complicated DNA nanostructure usually requires a high concentration of Na+or Mg2+to guarantee strong basepairing. More importantly, the as-synthesized silver nanoparticles appeared as a sharp band during agarose gel electrophoresis, which was extremely beneficial to the electrophoretic isolation of discrete DNA-silver nanoparticle conjugates. With the use of commercially available monothiolated DNA strands, valence-controllable DNA decoration of silver nanoparticles was realized for the first time. The silver nanoparticles bearing a specified number of DNA ligands were then purified through agarose gel electrophoresis. The as-obtained silver nanoparticle-DNA bionanoconjugates bearing a discrete number DNA ligands are ideal building blocks for DNA-programmable and valence-controlled self-assembly of nanostructured materials and functioning nanodevices. In addition, our experiments unambiguously revealed that a dual-thiol ligand could provide extra bonding strength for the silver nanoparticles, which will be important for research toward chemical and biological functionalizations of silver nanoparticles.The ability to monitor and regulate DNA hybridization kinetics on a nanoparticle’s surface is of great significance in many research areas including biosensors and DNA-based material assembly. Researchers have employed gold nanoparticles covered by a high density of DNA strands to investigate their hybridization kinetics, which, however, has various drawbacks that have to be taken into consideration. For example, the measured DNA coverage on a nanoparticle might be associated with significant errors due to multiple experimental steps. Also, the obtained DNA density was in fact a statistical average, instead of an accurate number as in the case of DNA monofunctionalized nanoparticles. Such hybridization reactions usually resulted in a wide distribution of hybridization products, and could be affected by some unexpected physical and chemical variables. Because DNA monofunctionalized nanoparticles are one of the most important construction units for DNA programmable nano-assembly, it would then be beneficial to use DNA monofunctionalized nanoparticles for the investigation of the hybridization kinetics of nanoparticle-tagged DNA sequences. We designed four different sets of DNA sequences for this investigation. After successful preparations of corresponding DNA monofunctionalized gold nanoparticles, the hybridization kinetics of single-stranded as well as partially double-stranded DNA ligands on gold nanoparticles were characterized and compared through gel electrophoretic analysis. Thanks to the simplicity of the reactants and products during the hybridization reactions, it was possible to extract hybridization rate constants by fitting the experimental data to a second order chemical reaction. The results revealed a relatively complicated hybridization kinetics of the nanoparticle-tagged DNA sequences, which was found to be highly sequence-dependent and temperature-dependent. This conclusion could be a useful guide for researchers who are seeking an efficient kinetic control for DNA-based nanoparticle assembly. In addition, our research also provided a useful technical platform for design and optimization of DNA sequences toward improved hybridization kinetics.Seed-strand initiated DNA self-assembly based on metastable DNA self-assembly modules has been used in mimicking dynamic chemical and biological reactions as well as in DNA nanomachinery. This potentially useful DNA self-assembly process (DNA self-polymerization) has been used for a fluorescence-based DNA sensor. However, electrochemical DNA sensing based on the DNA triggered self-assembly process has not been reported so far. Here we employed a seed-strand initiated DNA self-assembly process for the signal amplification of an electrochemical DNA sensor. The most prominent advantage of this strategy was that other materials such as proteins and nanoparticles were no longer needed for signal amplifications. Therefore, problems that are often associated with the use of enzymes and nanoparticles for electrochemical signal amplification, including strong electrode adsorptions, enzyme deactivations, high experimental cost, as well as nanoparticle aggregations, could be avoided. In this system, one hybridization event triggered the assembly of multiple DNA monomers, which significantly enhanced the electrochemical signal reflecting the existence of a DNA target. Besides, the assembly process was initiated from a surface-bound seed-strand and thus was restricted at the electrode/solution interface so that uncontrolled assembly of DNA monomers in the homogeneous solution was not possible. Under not yet optimized conditions, the detection limit of this assay was as low as4.9pM, which corresponded to a more than300-time improvement compared to an amplification-free system. The assay also exhibited excellent ability in differentiating a single base mutation within a DNA target. The strategy developed in this work should also be applicable to the detections of other important small and macromolecular targets (such as protein) besides DNA.