Tuning T4 DNA ligase action within self-assembled DNA nanostructures

The enzymatic ligation of DNA is a vital reaction in all living organisms, covalently joining fragmented DNA molecules during DNA replication, repair and recombination processes, and is fundamental for recombinant DNA technologies. Moreover, DNA nanotechnology has employed ligation reactions to increase the stability of DNA nanostructures. While it is known that T4 DNA ligase is hindered in DNA nanostructures, what mechanisms underpin its behaviour within a tightly packed system still needs to be understood. In response to this, the doctoral work presented here investigates the mechanism of action of T4 DNA ligase within a two-dimensional (2-D) triangular DNA origami formed by three identical trapezoids. The study was approached in two ways: first, the action of T4 DNA ligase within the inner, intertwine DNA molecules was investigated by focusing on the ligation of three consecutive staple strands within a trapezoid and analysing ligation products by using fluorescence labelling and gel electrophoresis. By assembling several variants of DNA triangle, including trapezoid pairs and monomers, and a much smaller DNA tile, it was discovered that the action of the T4 DNA ligase moderately depends on nanostructure mechanical properties. Indeed, the reaction efficiency can be modulated in a 100-fold range by varying nanostructure shape and size, with the latter parameter being essentially inversely proportional to ligation efficiency. The results suggest that the enzyme undergoes constrained diffusion in the DNA nanostructure. In the second study, the action of T4 DNA ligase was tested on linearised DNA segments in the gap between two flanking DNA origami triangles. The results obtained from gel electrophoresis, atomic force microscopy and nanopipette ionic current recording show that the reaction is hindered by the presence of the two flanking DNA nanostructures, despite the DNA substrate in much more accessible in solution than in the other study. Interestingly, the analysis in bulk proved more sensitive than the single molecule methods, which likely introduce higher disturbance to the nanostructure dimer. These unprecedented results will find applications in the synthesis of nucleic acid molecules with applications ranging from synthetic biology to DNA-based information storage devices.