Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA nanostructures in real time.

We describe a method for the real-time and high-throughput monitoring of the self-assembly and disassembly of complex DNA superstructures, using temperature-dependent Förster resonance energy transfer (FRET) spectroscopy. Compared with other spectroscopic approaches, such as UV-visible and circular dichroism, the method described has advantages in terms of sensitivity, feasibility for high-throughput analysis and applicability to virtually any kind of supramolecular structure. To this end, two oligonucleotides out of the entire set building up the superstructure are labeled with a fluorescein and a tetramethylrhodamine, as FRET donor and acceptor, respectively. Correct assembly of the superstructure induces maximum FRET efficiency, whereas complete dissociation leads to minimal FRET. Monitoring of temperature-dependent FRET efficiency yields a thermal profile that is used for thermodynamic analysis. In the case of reversible and cooperative assembly/disassembly of the DNA superstructure, application of the van't Hoff law allows for the determination of the thermodynamic parameters of the process. Owing to slow temperature ramping, the entire assay requires about 17 h. The protocol allows to simultaneously analyze up to 384 samples with only 30 microl sample volume each.