Concatemerization of Synthetic Oligonucleotides
Doctoral thesis, 2014
DNA nanotechnology has become an important research field because of its advantages in high predictability and accuracy of base-paring recognition. For example, the DNA concatemer, one of the simplest DNA constructs in shape, has been used to enhance signals in biosensing. In this thesis, concatemers were designed and characterized towards sequence-specific target amplifiers for single molecule mechanical studies.
The project firstly focuses on exploring how to obtain concatemers of satisfactory length by self-assembly in bulk. Concatemers formed in solution by mixing of the different components were characterized by gel electrophoresis and AFM. Experimental results show that the concatemer yield could be increased primarily by increasing the ionic strength, and linear concatemers of expected length could be separated from mixed sizes and shapes. As an alternative, a platform for concatemer formation based on a planar surface was investigated by immobilizing and sequentially hybridizing oligonucleotides to concatemers. This method was further improved by involving click-reaction to link the nicks on backbones of the concatemers. 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.
Furthermore, the surface-based concatemer formation platform was applied to study DNA-ligand interaction using QCM-D and SPR. We selected three ligands, known to bind to DNA: spermidine, spermine and RAD51. We observed that polyamines condense the DNA layers; by contrast, RAD51 induces an extension of the DNA layer. This study provides some guidelines for QCM-D and SPR-based characterization of changes in the properties of DNA films (e.g., thickness, viscoelastic properties) on sensor surfaces. It is interesting to correlate these changes to structural and mechanical changes induced in single DNA molecules upon ligand binding. The biosensing approach provides rapid access to kinetic data of DNA-ligand interactions, which is typically useful in various screening applications.