Analysis of the Self-Assembly of 4 x 4 DNA Tiles by Temperature-Dependent FRET Spectroscopy

Correct and efficient self-assembly of oligonucleotides into highly ordered superstructures essentially depends on the structural integrity and thermal stability of DNA motifs such as junctions or tiles that build up the superstructure. To investigate the assembly/disassembly process of DNA tiles, we recently described a microplate-based method employing Forster resonance energy transfer (FRET) spectroscopy, which enables the analysis of DNA superstructure formation in real time and with high throughput. This method allows thermodynamic parameters of the self-assembly process to be extracted, and we here apply it for detailed analysis of the self-assembly of five different 4x4 DNA tile motifs. To specifically investigate whether the FRET probes tethered to the DNA motifs report local thermodynamic stabilities in the immediate proximity of the chromophores, or whether the global stability of the entire motif is monitored, systematic variations of the labeling position within one tile are carried out. Combined with gel electrophoretic, UV spectroscopic, and microcalorimetric analysis, this study reveals that the FRET method mainly reports the thermodynamics of local microenvironment assembly, rather than that of the entire motif. Nonetheless, the thermodynamic data derived from FRET analysis are also influenced by the structural surroundings of the motif, and thus rapid and detailed analysis and identification of potential weak points within a superstructure which influence the structural integrity of a given tile design are enabled. Therefore, the microplate FRET method readily provides insights into the assembly process of complex DNA superstructures to verify and complement theoretical design approaches.