DNA nanostructure decoration: a how-to tutorial.

DNA Nanotechnology is being applied to multiple research fields. The functionality of DNA nanostructures is significantly enhanced by decorating them with nanoscale moieties including: proteins, metallic nanoparticles, quantum dots, and chromophores. Decoration is a complex process and developing protocols for reliable attachment routinely requires extensive trial and error. Additionally, the granular nature of scientific communication makes it difficult to discern general principles in DNA nanostructure decoration. This tutorial is a guidebook designed to minimize experimental bottlenecks and avoid dead-ends for those wishing to decorate DNA nanostructures. We supplement the reference material on available technical tools and procedures with a conceptual framework required to make efficient and effective decisions in the lab. Together these resources should aid both the novice and the expert to develop and execute a rapid, reliable decoration protocols.

Analysis of DNA Origami Nanostructures Using Capillary Electrophoresis.

DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (Rs = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (As = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.

Binding, brightness, or noise? Extracting temperature-dependent properties of dye bound to DNA.

We present a method for extracting temperature-dependent thermodynamic and photophysical properties of SYTO-13 dye bound to DNA from fluorescence measurements. Together, mathematical modeling, control experiments, and numerical optimization enable dye binding strength, dye brightness, and experimental noise (or error) to be discriminated from one another. By focusing on the low-dye-coverage regime, the model avoids bias and can simplify quantification. Utilizing the temperature-cycling capabilities and multi-reaction chambers of a real-time PCR machine increases throughput. Significant well-to-well and plate-to-plate variation is quantified by using total least squares to account for error in both fluorescence and nominal dye concentration. Properties computed independently for single-stranded DNA and double-stranded DNA by numerical optimization are consistent with intuition and explain the advantageous performance of SYTO-13 in high-resolution melting and real-time PCR assays. Distinguishing between binding, brightness, and noise also clarifies the mechanism for the increased fluorescence of dye in a solution of double-stranded DNA compared to single-stranded DNA; in fact, the explanation changes with temperature.

Synthesizing the biochemical and semiconductor worlds: the future of nucleic acid nanotechnology.

Since its inception nearly 40 years ago [Kallenbach, et al., Nature, 1983, 305, 829; N. C. Seeman, J. Theoretical Biology, 1982, 99, 237], Nucleic Acid Nanotechnology (NAN) has matured and is beginning to find commercial applications. For the last 20 years, it has been suggested that NAN might be an effective replacement for parts of the semiconductor lithography or protein engineering workflows. However, in that time, these incumbent technologies have made significant advances, and our understanding of NAN's strengths and weaknesses has progressed, suggesting that the greatest opportunities in fact lie elsewhere. Given the commitment of resources necessary to bring new technologies to the market and the desire to use those resources as wisely as possible, we conduct a critical examination of where NAN may benefit from, and provide benefit to, adjacent technologies and compete least with market incumbents. While the accuracy of our conclusions may be limited by our ability to extrapolate from the current state of NAN to its future commercial success, we conclude that the next promising direction is to create a bridge between biology and semiconductor technology. We also hope to stimulate a robust conversation around this technology's capabilities with the goal of building consensus on those research and development efforts that would advance NAN to the greatest effect in real-world applications.

Best practice for improved accuracy: A critical reassessment of van't Hoff analysis of melt curves.

Biomolecular thermodynamics, particularly for DNA, are frequently determined via van't Hoff analysis of optically measured melt curves. Accurate and precise values of thermodynamic parameters are essential for the modeling of complex systems involving cooperative effects, such as RNA tertiary structure and DNA origami, because the uncertainties associated with each motif in a folding energy landscape can compound, significantly reducing the power of predictive models. We follow the sources of uncertainty as they propagate through a typical van't Hoff analysis to derive best practices for melt experiments and subsequent data analysis, assuming perfect signal baseline correction. With appropriately designed experiments and analysis, a van't Hoff approach can provide surprisingly high precision, e.g., enthalpies may be determined with a precision as low as 10-2 kJ ⋅ mol-1 for an 8-base DNA oligomer.

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

Understanding the folding process of DNA origami is a critical stepping stone to the broader implementation of nucleic acid nanofabrication technology but is notably nontrivial. Origami are formed by several hundred cooperative hybridization events-folds-between spatially separate domains of a scaffold, derived from a viral genome, and oligomeric staples. Individual events are difficult to detect. Here, we present a real-time probe of the unit operation of origami assembly, a single fold, across the scaffold as a function of hybridization domain separation-fold distance-and staple/scaffold ratio. This approach to the folding problem elucidates a predicted but previously unobserved blocked state that acts as a limit on yield for single folds, which may manifest as a barrier in whole origami assembly.

Analysis and uncertainty quantification of DNA fluorescence melt data: Applications of affine transformations.

Fluorescence-based measurements are a standard tool for characterizing the thermodynamic properties of DNA systems. Nonetheless, experimental melt data obtained from polymerase chain-reaction (PCR) machines (for example) often leads to signals that vary significantly between datasets. In many cases, this lack of reproducibility has led to difficulties in analyzing results and computing reasonable uncertainty estimates. To address this problem, we propose a data analysis procedure based on constrained, convex optimization of affine transformations, which can determine when and how melt curves collapse onto one another. A key aspect of this approach is its ability to provide a reproducible and more objective measure of whether a collection of datasets yields a consistent "universal" signal according to an appropriate model of the raw signals. Importantly, integrating this validation step into the analysis hardens the measurement protocol by allowing one to identify experimental conditions and/or modeling assumptions that may corrupt a measurement. Moreover, this robustness facilitates extraction of thermodynamic information at no additional cost in experimental time. We illustrate and test our approach on experiments of Förster resonance energy transfer (FRET) pairs used study the thermodynamics of DNA loops.

Revealing thermodynamics of DNA origami folding via affine transformations.

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.

pH-Driven Actuation of DNA Origami via Parallel I-Motif Sequences in Solution and on Surfaces.

As bottom up DNA nanofabrication creates increasingly complex and dynamic mechanisms, the implementation of actuators within the DNA nanotechnology toolkit has grown increasingly important. One such actuator, the I-motif, is fairly simple in that it consists solely of standard DNA sequences and does not require any modification chemistry or special purification beyond that typical for DNA oligomer synthesis. This study presents a new implementation of parallel I-motif actuators, emphasizing their future potential as drivers of complex internal motion between substructures. Here we characterize internal motion between DNA origami substructures via AFM and image analysis. Such parallel I-motif design and quantification of actuation provide a useful step toward more complex and effective molecular machines.