Revealing thermodynamics of DNA origami folding via affine transformations

Abstract

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, 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 potential secondary and tertiary structure of the scaffold. Our approach also highlights the importance of heat-capacity changes during DNA melting. In addition to yielding insight into DNA origami folding, it is well-suited to probing fundamental processes in related self-assembling systems.

Failure Mechanisms in DNA Self-Assembly: Barriers to Single-Fold Yield

“…, <1), this assumption may be less robust when the folding process incurs a large entropic penalty. Our system comprises the scaffold molecule, a folding oligomer staple, and two reporter oligomers labeled with a fluorophore and quencher, respectively . The scaffold consists of the M13mp18 genome hybridized over most of its length, with long oligomers designed to remove secondary structure, as discussed in the Methods.…”

“…The reporter shown in Figure b signals a change in topology rather than a change in hybridization. Using our previously developed , affine transformation technique to improve background correction, together with approximate knowledge of the fluorescence of the quenched system, we estimate the fraction of the scaffold in the folded and blocked states. This simple reporting scheme is only viable due to the reduced nature of the system: a typical staple with three domains would require at least five reporting channels.…”

“…In this work, we use a variation of the hierarchical affine transformation data analysis technique developed previously. , This technique assumes that every measured data set is generated by a “true” signal, σ­( T ), that is distorted by an unknown amount of temperature-dependent fluorescence and background effects. For example, we model the DNA measurements presented here, using eq , where m i ( T ) is the i th measurement, R ( T ) is a temperature-dependent fluorescence efficiency, B ( T ) is a background signal, and the unknown affine parameters, a i , b i , and c i , characterize the relative contributions of each source.…”

“…The occurrence of any given fold influences the kinetics and thermodynamics of the remaining folds by changing the stiffness and distances between binding domains of those folds. In other words, the configurational entropy associated with binding the scaffold acts as an energetic penalty collectively across all binding events that change the topology. ,, …”

Insights into the Structure and Energy of DNA Nanoassemblies

“…Other theories have been advanced that build on this minimalist approach and consider additional contributions to explain the observed cooperativity of DNA origami folding, as well as the asymmetry of its hysteresis. Such contributions include the coaxial stacking of adjacent domains and Kuhn lengths approximations of the loop, as well as a conformational entropy and binding enthalpy that affect the step-wise folding of DNA origami structures in a loop’s length-dependent fashion [ 96 , 97 ].…”