A mean-field theory for characterizing the closing rates of DNA origami hinges.

The evolution of dynamic DNA nanostructures has propelled DNA nanotechnology into a robust and versatile field, offering groundbreaking applications in nanoscale communication, drug delivery, and molecular computing. Yet, the full potential of this technology awaits further enhancement through optimization of kinetic properties governing conformational changes. In this work, we introduce a mean-field theory to characterize the kinetic behavior of a dynamic DNA origami hinge where each arm bears complementary single-stranded DNA overhangs of different lengths, which can latch the hinge at a closed conformation. This device is currently being investigated for multiple applications, being of particular interest the development of DNA-based rapid diagnostic tests for coronavirus. Drawing from classical statistical mechanics theories, we derive analytical expressions for the mean binding time of these overhangs within a constant hinge. This analysis is then extended to flexible hinges, where the angle diffuses within a predetermined energy landscape. We validate our model by comparing it with experimental measurements of the closing rates of DNA nanocalipers with different energy landscapes and overhang lengths, demonstrating excellent agreement and suggesting fast angular relaxation relative to binding. These findings offer insights that can guide the optimization of devices for specific state lifetimes. Moreover, the framework introduced here lays the groundwork for further advancements in modeling the kinetics of dynamic DNA nanostructures.

Designed architectural proteins that tune DNA looping in bacteria.

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

DNA simulation benchmarks revealed with the accumulation of high-resolution structures.

DNA carries more than the list of biochemical instructions that drive the basic functions of living systems. The sequence of base pairs includes a multitude of structural and energetic signals that determine the degree to which the long, threadlike molecule moves and how it responds to proteins and other molecules involved in its processing and packaging. The arrangements of successive base pairs in high-resolution protein-DNA crystal structures provide useful benchmarks for atomic-level simulations of double-helical DNA as well as information potentially useful in interpreting the properties of specific DNA sequences. The set of currently available structures has enough examples to characterize the conformational preferences of the DNA base-pair steps within the context of their immediate neighbors, i.e., in the context of tetramers, and reveals surprising effects of certain neighbors on local chain properties. The proteins in contact with DNA present various microenvironments that sense and/or induce the observed spatial forms. The cumulative buildup of amino-acid atoms in different protein-DNA complexes produces a binding cloud around the double helix with subtle sequence-dependent features. While the microenvironment presented by each protein to DNA is highly unique, the overall composition of amino-acid atoms within close range of DNA in a broad collection of structures is fairly uniform. The buildup of protein atoms of different types around the DNA provides new information for the improvement of nucleic acid force fields and fresh ideas for the exploration of the properties of DNA in solution.

Optimizing Binding among Bimolecular Tethered Complexes.

Tethered motion is ubiquitous in nature, offering controlled movement and spatial constraints to otherwise chaotic systems. The enhanced functionality and practical utility of tethers has been exploited in biotechnology, catalyzing the design of novel biosensors and molecular assembly techniques. While notable technological advances incorporating tethered motifs have been made, a theoretical gap persists within the paradigm, hindering a comprehensive understanding of tethered-based technologies. In this work, we focus on the characterization of the binding kinetics of two tethered molecules functionalized to a hard surface. Using a mean-field approximation, the binding time of such bimolecular system is determined analytically. Furthermore, estimates of the grafting site separation and polymer lengths which expedite binding are provided. These estimates, along with the analytical theories and frameworks established here, have the potential to improve efficacy in self-assembly methods in DNA nanotechnology and can be extended to more biologically specific endeavors including targeted drug-delivery and molecular sensing.

emDNA - A Tool for Modeling Protein-decorated DNA Loops and Minicircles at the Base-pair Step Level.

Computational modeling of nucleic acids plays an important role in molecular biology, enhancing our general understanding of the relationship between structure and function. Biophysical studies have provided a wealth of information on how double-helical DNA responds to proteins and other molecules in its local environment but far less understanding of the larger scale structural responses found in protein-decorated loops and minicircles. Current computational models of DNA range from detailed all-atom molecular dynamics studies, which produce rich time and spatially dependent depictions of small DNA fragments, to coarse-grained simulations, which sacrifice detailed physical and chemical information to treat larger-scale systems. The treatment of DNA used here, at the base-pair step level with rigid-body parameters, allows one to develop models hundreds of base pairs long from local, sequence-specific features found from experiment. The emDNA software takes advantage of this framework, producing optimized structures of DNA at thermal equilibrium with built-in or user-generated elastic models. The program, in combination with the case studies included in this article, allows users of any skill level to develop and investigate mesoscale models of their own design. The functionality of emDNA includes a tool to incorporate experiment-specific configurations, e.g., protein-bound and/or melted DNA from known high-resolution structures, within higher-order 3D models by fixing the orientation and position of user-specified base pairs. The software provides a new avenue into multiscale genetic modeling, giving a wide range of users a deeper understanding of DNA mesoscale organization and the opportunity to pose new questions in genetic research. The publicly available emDNA software, including build instructions and usage information, is available on GitHub (https://nicocvn.github.io/emDNA/).

Revisiting DNA Sequence-Dependent Deformability in High-Resolution Structures: Effects of Flanking Base Pairs on Dinucleotide Morphology and Global Chain Configuration.

DNA carries more than the list of biochemical ingredients that drive the basic functions of living systems. The sequence of base pairs includes a multitude of structural and energetic signals, which determine the degree to which the long, threadlike molecule moves and how it responds to proteins and other molecules that control its processing and govern its packaging. The chemical composition of base pairs directs the spatial disposition and fluctuations of successive residues. The observed arrangements of these moieties in high-resolution protein-DNA crystal structures provide one of the best available estimates of the natural, sequence-dependent structure and deformability of the double-helical molecule. Here, we update the set of knowledge-based elastic potentials designed to describe the observed equilibrium structures and configurational fluctuations of the ten unique base-pair steps. The large number of currently available structures makes it possible to characterize the configurational preferences of the DNA base-pair steps within the context of their immediate neighbors, i.e., tetrameric context. Use of these knowledge-based potentials shows promise in accounting for known effects of sequence in long chain molecules, e.g., the degree of curvature reported in classic gel mobility studies and the recently reported sequence-dependent responses of supercoiled minicircles to nuclease cleavage.

Surprising Twists in Nucleosomal DNA with Implication for Higher-order Folding.

While nucleosomes are dynamic entities that must undergo structural deformations to perform their functions, the general view from available high-resolution structures is a largely static one. Even though numerous examples of twist defects have been documented, the DNA wrapped around the histone core is generally thought to be overtwisted. Analysis of available high-resolution structures from the Protein Data Bank reveals a heterogeneous distribution of twist along the nucleosomal DNA, with clear patterns that are consistent with the literature, and a significant fraction of structures that are undertwisted. The subtle differences in nucleosomal DNA folding, which extend beyond twist, have implications for nucleosome disassembly and modeled higher-order structures. Simulations of oligonucleosome arrays built with undertwisted models behave very differently from those constructed from overtwisted models, in terms of compaction and inter-nucleosome contacts, introducing configurational changes equivalent to those associated with 2-3 base-pair changes in nucleosome spacing. Differences in the nucleosomal DNA pathway, which underlie the way that DNA enters and exits the nucleosome, give rise to different nucleosome-decorated minicircles and affect the topological mix of configurational states.