Structural DNA nanotechnology, as exemplified by DNA origami, has enabled the design and construction of molecularly precise objects for a myriad of applications. However, limitations in imaging, and other characterization approaches, make a quantitative understanding of the folding process challenging. Such an understanding is necessary to determine the origins of structural defects, and hence function, which constrain the practical use of these nanostructures. Here, we combine careful fluorescent reporter design with a novel affine transformation technique that together permit the rigorous measurement of folding thermodynamics. This method removes sources of systematic uncertainty and resolves problems with typical background correction schemes. This in turn allows us to examine entropic corrections associated with folding and the secondary and tertiary structure of the scaffold. In addition, the approach highlights the role of heat capacity changes in DNA melting. In addition to yielding insight into DNA origami folding, our is well-suited to probing fundamental processes in related self-assembling systems.
Nucleic Acids Research
DNA, thermodynamics, configurational entropy, dna origami, melt curves