Concatemerization of Synthetic Oligonucleotides, Assembly on Surface and Ligand Interactions
Doctoral thesis, 2014
DNA nanotechnology has become an important research field because of its advantages in high predictability and accuracy of base-paring recognition. DNA concatemer, one of the simplest DNA constructs in shape, have been found as efficient signal amplifiers in several biosensors for different applications. In this thesis, formation of concatemers as sequence-specific target amplifier for single molecular mechanical study was constructed and characterized.
The research work firstly focuses on exploring to obtain concatemers in satisfactory length by self-assembled method via varying incubation conditions. Formed concatemers were characterized by gel electrophoresis and AFM. Experimental results show that the efficiency of forming concatemers could be optimized primarily by increasing ionic strength. Moreover, linear concatemers with expected length could be separated from mixed sizes and shapes.
In the second part, an attempt of establishing a platform on planar surface is investigated by immobilizing and sequentially hybridizing oligonucleotides to concatemers. This method is further improved by involving click-reaction to link the nicks on the backbones. QCM-D and SPR were utilized to monitor the step by step construction process. Gel electrophoresis shows that concatemers with desired length were successfully formed in a controllable manner.
Finally, we apply the concatemer formation platform to study DNA-ligand interaction using QCM-D. We selected three ligands to bind to DNA: spermidine, spermine and RAD51. We observe that polyamines condense the DNA layers; by contrast, RAD51 induces an extension of the DNA layer. This study provides some guidelines for a QCM-D biosensor based on ligand-induced structural changes of DNA films.