Session A29: DNA-based Soft Matter I

Effect of localized active fluctuations in conformation and dynamics of chromosomal DNA

Active fluctuations play a significant role in the structure and dynamics of biopolymers that are instrumental in the functioning of living cells, including the proteins and nucleic acids that comprise chromosomal DNA. For a large range of experimentally accessible length and time scales, chromosomal DNA subject to varying transcriptional and topoisomerase activity can be represented as a flexible chain that is subject to active fluctuations that depends on genomic position. In this work, we introduce a mathematical framework that integrates the spatial and temporal patterns of the fluctuations on a flexible polymer to different observables that describe the dynamics and conformations of the polymer. We identify the length and time scale where the behavior of the polymer takes a significant departure from the behavior of an equilibrium chain. Furthermore, we show that the localized nature of fluctuation introduces a distinction of behavior between the segments near and far from the source of the fluctuation both in position and time. We then consolidate these predictions into a framework to interpret the active-force distribution from experimental measurements of chromosomal dynamics. Altogether, this work sets the basis for understanding and interpreting the role of spatiotemporal pattern of fluctuations in the dynamics, conformation, and eventually the functionality of the biopolymers such as chromosomes in living cells and in vitro DNA subject to varying biological processes.   

microtubule self-assembly is controlled by the topological activity of ring and linear DNA in microtubule-DNA composites

Polymer composites, ubiquitous in biological systems, display complex behaviors influenced by the topology of their constituent components. For example, we previously found that the polymerization of tubulin into microtubules was markedly suppressed when embedded in an entangled solution of ring DNA, whereas the same concentration of linear DNA led to enhanced polymerization and flocculation of microtubules, resulting in a non-monotonic dependence of composite stiffness on tubulin concentration. Here, we investigate the time-dependent structural and mechanical properties of similar DNA-microtubule composites, undergoing in situ enzymatically-driven linearization and fragmentation of DNA rings, to determine the impact of DNA digestion on the self-assembly of the microtubule network. We use Optical Tweezers integrating Differential Dynamic Microscopy (OpTiDMM) to measure the time-dependent rheological properties and couple rheology to the changing structure and dynamics of the microtubules and DNA in the networks. Our preliminary results indicate that in situ topological conversion significantly alters the structural evolution of the microtubule networks, with DNA fragmentation promoting flocculation at a rate controlled by the enzyme stoichiometry. This topologically-active composite demonstrates a novel approach to controlling the kinetics of network self-assembly, as well as the initial and final mechanical and structure states, by performing topological operations on the surrounding polymers.   

Effective interactions between double-stranded DNA molecules in aqueous electrolyte solutions: effects of molecular architecture and counterion valency

Spontaneous self-assembly of DNA molecules is ubiquitous in biological systems and DNA-DNA interactions are relevant to numerous biological processes, including genome compaction, and homologous recombination, as well as in emerging DNA nanotechnological applications, such as functionalized DNA origami, as well as natural and synthesized DNA-catenane structures [1]. Complementary to experimental and theoretical studies, simulations offer an appealing alternative route for the study of these systems at an atomistic level, allowing for the detailed observation of the microscopic molecular conformation of DNA and the ionic atmosphere. The scope of the work presented here is the computational investigation of the effects of DNA molecular topology, namely linear and circular, as well as counterion valency, on the ensuing pairwise effective interactions between DNA molecules in an un-linked and linked (catenated) state. Umbrella sampling simulations were performed, and effective potentials have been computed by employing the weighted histogram analysis method. An interesting comparison can be drawn between the different DNA topologies studied here, regarding the contrasting effects of divalent counterions on the effective potentials, and this effect can be attributed to the fact that linear DNA fragments can easily adopt relative orientations that minimize electrostatic and steric repulsions by rotating relative to one another and by exhibiting more pronounced bending.   

The underappreciated role of nonspecific attractions in crystallization of DNA-coated colloids

 DNA-coated colloids are ideal building blocks for studying self assembly due to the relative ease in programming chemically specific interactions between the constituent particles. The effective interactions that emerge are often modeled as having a short range attraction from DNA hybridization and a short range repulsion from the polymer and/or DNA brush on the surface of the particles. Furthermore, it is often assumed that these are the only relevant interactions that dictate the assembly of a wide variety of crystalline structures. In this talk, I will show that other non-specific attractive interactions do in fact alter the structure of DNA-programmed colloidal crystals and how changes in the particle synthesis can tune the strength of non-specific interactions. For example, I will show that the structure of an assembled crystal phase can be altered by adjusting the length of an inert polymer grafted to the particle surface, which we attribute to a competition between Van der Waals attraction and steric repulsion. This research indicates that the phase behavior of DNA-coated colloids is more complex than typically assumed and that nonspecific interactions can play a relevant role in determining the types of crystalline structures that form.   

SAT-assembly: A method for model-driven inverse design of self-assembling 3D lattices, capsids, and polycubes

The control over the self-assembly of complex structures is a long-standing challenge of material science, especially at the colloidal scale, as the desired assembly pathway is often kinetically derailed by the formation of competing alternative structures or amorphous aggregates. The goal of inverse design problem is hence to find a set of blocks that reliably assemble in high yield into a target structure while avoiding kinetic traps and alternative competing states. We present here design method

“SAT-assembly”, which solves this problem by mapping the design problem in terms of different building block types and interactions between them to Boolean Satisfiability problem (SAT). We then show examples of application of our design pipeline to several highly sought-after self-assembly designs. We first study the design of self-assembled capsids.We next compare different design strategies for finite-size polycube structures built from cube-shaped subunits. Finally, we design colloidal system that self-assemble pyrochlore lattices that are of interest for optical metamaterial construction. For each studied system, we demonstrate its experimental realization by self-assembling DNA origami nanostructures.   

Sliding tubules: adding additional degrees of freedom to anneal self-assembled DNA origami structures

Self-assembly of structures that have complex geometries, such as open crystalline lattices, shells, or tubules, requires subunits with valence-limited interactions to correctly orient the subunits within the structure. However, valence-limited interactions can limit subunit mobility within an assembly, preventing them from annealing out of off-target structures. Introducing new degrees of freedom can alleviate these kinetic traps. In this talk, I will describe how we apply these ideas to reduce polymorphism in self-assembled tubules. We assemble tubules from DNA origami subunits with specific interactions and binding angles. Despite targeting a single tubule geometry, thermal fluctuations allow a variety of tubule types to form. Since subunits lack mobility, tubules cannot relax to equilibrium even as they grow and accumulate internal elastic stress. To overcome this challenge, we tweak the interactions to create sliding modes within the assemblies. We find that these new modes allow the tubules to anneal towards the target structure. These results show how including additional degrees of freedom can promote annealing towards equilibrium, thereby reducing polymorphism as we strive to make more complex structures.   

Scaling down bioreactor processes to create unimolecular supercoiled, cyclic, and linear DNA for study of topology effects in soft materials.

Biomass DNA materials have recently gained traction due to declining DNA costs and their relatively underexplored applications at bulk scales. Due to the demand for nucleic acid therapeutics, bioreactor based production has tremendously increased the yield of plasmid DNA to > 1 g of purified plasmid DNA (pDNA) per liter of culture fluid. In this work we discuss reverse engineering industrial scale fermentation and purification strategies to access gram scale amounts of pDNA which will act as precursors to DNA hydrogel materials with controlled topological elements. Beyond its secondary structure, dsDNA exhibits exquisite variety in its geometric shape and topological form, but we have not yet tapped the full potential of this feature as a tool to fine-tune and control the properties of materials on a molecular scale. As these topological forms influence virtually every DNA process, vast literature precedent dedicated to understanding the role of DNA topology in life processes would serve as the ideal foundation for a unique material. We report up to 100 mg/mL solutions of unimolecular DNA.   

Deformation of Kinetoplast DNA in Microfluidic Racetracks

The discovery of graphene opened the door to studying many types of two-dimensional crystalline materials, but two-dimensional soft materials have also garnered interest. To find a suitable model for soft two-dimensional polymers, we have studied the properties of kinetoplast DNA (kDNA). Found in the mitochondria of the trypanosome parasite, kDNA are comprised of topologically interconnected small loops of DNA which create an effectively two-dimensional catenated network. In this work we examine the flow-induced deformation in kinetoplast as they translate through a microfluidic racetrack. Pressure-driven flow induces shear forces on the molecules, whereas electrophoretic flow may deform the molecule in regions of non-uniform electric field.  We examined the anisotropy of the kDNA as it flowed thought the micro channels under flow and electrophoresiss. We discuss results relating the flow-induced deformation to the size and speed of the translating networks.   

Single-molecule dynamics of tethered DNA in shear flow: the effect of viscosity

Deoxyribonucleic acid (DNA) is a highly charged, semiflexible polymer. Chromosomal DNA is much longer than cell dimensions, and various DNA-binding proteins are involved in compacting and organizing chromosomal DNA in the tiny volume of the cell or nucleus. Single-molecule DNA flow-stretching is a widely employed, powerful technique of investigating the underlying mechanisms of these DNA-binding proteins. Here, we combine experiment and simulation to study the effect of buffer viscosity on DNA flow-stretching and DNA fluctuations. Surface-tethered bacteriophage lambda DNA was stretched by hydrodynamic drag force in a flow cell, and the positions of the free end of the DNA were recorded in real time by tracking a quantum dot labeled at the free end. We found that an increase of buffer viscosity results in an increase of DNA length and a decrease of fluctuations of the freely moving end of the DNA. To better understand our experimental results, we performed extensive Brownian dynamics simulations of a bead-spring chain model of the DNA. Static and dynamic properties of the DNA such as the end-to-end distance and correlation functions were determined as a function of the Weissenberg number, Wi, and the relaxation time, τ, of the polymer. Our simulations agree well with our experimental results for the buffer viscosity.   

Characterizing the nucleation and crystal growth of DNA origami nanoparticles

DNA origami is a powerful technology to manipulate various self-assembled structures by programming the specific interactions and complex shapes of nanoscale subunits. Though much effort has focused on controlling the final structures of different self-assembled crystal phases, studying their growth dynamics remains challenging due to the fact that the subunits are much smaller than the wavelength of visible light. In this talk, I will describe our experimental approach to quantifying the nucleation and growth of crystals formed from octahedral DNA origami nanoparticles.In particular, we study crystallization of DNA origami confined to monodisperse water-in-oil droplets made via microfluidics. Inside the droplets, the nucleation and crystal growth are monitored as a function of time to extract nucleation rates and growth laws despite the fact that we lack single-particle resolution. We use the same platform to compare the growth dynamics of different crystal symmetries that can be assembled from the same subunits by changing the aqueous conditions. We expect that these results will advance the understanding of the dynamics and physical properties of nucleation and crystal growth of nanometer-size subunits.   

Force-induced structural transitions in DNA nanostructures

Mechanical properties of biomolecules provide valuable insights into their structural characteristics and translation to applications. Deformation modes and rigidity can be used to characterize soft objects such as DNA, exosomes, and liposomes. With the advent of DNA nanotechnology, DNA has been increasingly used as a key component of soft programmable materials. Among the many advantages of the technology, the ability to define and control the mechanical properties of these materials is enabling. However, the deformation of DNA nanostructures subject to forces remains underexplored. 

Coarse-grained (CGMD) DNA simulations are a valuable tool to probe the mechanical response of these materials. In this study, we investigate the deformation of the building blocks of DNA nanostructures, linear dsDNA chains and circular rings, in elongational flow. We observe structural transitions in the DNA conformation which are otherwise beyond the resolution of experimental techniques. These transitions, related to overstretching and hyperstretching, are accompanied by increases in the DNA base-pair distances due to coordinated unwinding and force-induced melting. 

Using CGMD simulations, we elucidate the role of the local tension force, DNA sequence and energy barrier in eliciting different structural transitions of DNA in flow. Our work provides valuable insights into the nonlinear elastic response regimes and structural conformations of DNA nanostructures that have not been previously probed.   

DNA-Functionalized Nanoparticles in Mixed Electrolytes: Salting In, Out, and Beyond

Dissolved ions mediate interactions between charged biomolecules. While general principles for the effects of added salts on proteins have been established, comprehensive descriptions have yet to be achieved for DNA-based nanomaterials. Here, we analyze how interactions between nanoparticles grafted with non-base-pairing DNA change with the composition of the surrounding electrolyte. Small-angle X-ray scattering measurements reveal that specific cations (e.g., Ca²⁺) drive the assembly of colloidal crystals. At low salinity, the addition of NaCl suppresses this crystallization, consistent with “salting in” mechanism, where NaCl screens attractive electrostatic interactions. At higher salt concentrations, crystallization instead becomes increasingly favorable, suggesting a transition to a “salting out” regime, where added salt dehydrates the DNA. At extremely high salinity, DNA-NP aggregates swell with added salt, reflecting an additional transition to a concentrated electrolyte regime. The roles of solvent, anions, and temperature will be discussed. These results demonstrate analogous behavior of proteins and DNA-NPs, and also outline additional design considerations for DNA-based materials.