DNA Brushes Research Articles

Controlling Silicification on DNA Origami with Polynucleotide Brushes.

DNA origami has been used as biotemplates for growing a range of inorganic materials to create novel organic-inorganic hybrid nanomaterials. Recently, the solution-based silicification of DNA has been used to grow thin silica shells on DNA origami. However, the silicification reaction is sensitive to the reaction conditions and often results in uncontrolled DNA origami aggregation, especially when growth of thicker silica layers is desired. Here, we investigated how site-specifically placed polynucleotide brushes influence the silicification of DNA origami. Our experiments showed that long DNA brushes, in the form of single- or double-stranded DNA, significantly suppress the aggregation of DNA origami during the silicification process. Furthermore, we found that double-stranded DNA brushes selectively promote silica growth on DNA origami surfaces. These observations were supported and explained by coarse-grained molecular dynamics simulations. This work provides new insights into our understanding of the silicification process on DNA and provides a powerful toolset for the development of novel DNA-based organic-inorganic nanomaterials.

Computational analysis of protein synthesis, diffusion, and binding in compartmental biochips

Protein complex assembly facilitates the combination of individual protein subunits into functional entities, and thus plays a crucial role in biology and biotechnology. Recently, we developed quasi-twodimensional, silicon-based compartmental biochips that are designed to study and administer the synthesis and assembly of protein complexes. At these biochips, individual protein subunits are synthesized from locally confined high-density DNA brushes and are captured on the chip surface by molecular traps. Here, we investigate single-gene versions of our quasi-twodimensional synthesis systems and introduce the trap-binding efficiency to characterize their performance. We show by mathematical and computational modeling how a finite trap density determines the dynamics of protein-trap binding and identify three distinct regimes of the trap-binding efficiency. We systematically study how protein-trap binding is governed by the system’s three key parameters, which are the synthesis rate, the diffusion constant and the trap-binding affinity of the expressed protein. In addition, we describe how spatially differential patterns of traps modulate the protein-trap binding dynamics. In this way, we extend the theoretical knowledge base for synthesis, diffusion, and binding in compartmental systems, which helps to achieve better control of directed molecular self-assembly required for the fabrication of nanomachines for synthetic biology applications or nanotechnological purposes.

Tuning the Effective Interactions between Spherical Double-Stranded DNA Brushes

Tuning the Effective Interactions between Spherical Double-Stranded DNA Brushes

Blunt-End Driven Re-entrant Ordering in Quasi Two-Dimensional Dispersions of Spherical DNA Brushes.

We investigate the effects of crowding on the conformations and assembly of confined, highly charged, and thick polyelectrolyte brushes in the osmotic regime. Particle tracking experiments on increasingly dense suspensions of colloids coated with ultralong double-stranded DNA (dsDNA) fragments reveal nonmonotonic particle shrinking, aggregation, and re-entrant ordering. Theory and simulations show that aggregation and re-entrant ordering arise from the combined effect of shrinking, which is induced by the osmotic pressure exerted by the counterions absorbed in neighbor brushes and of a short-range attractive interaction competing with electrostatic repulsion. An unconventional mechanism gives origin to the short-range attraction: blunt-end interactions between stretched dsDNA fragments of neighboring brushes, which become sufficiently intense for dense and packed brushes. The attraction can be tuned by inducing free-end backfolding through the addition of monovalent salt. Our results show that base stacking is a mode parallel to hybridization to steer colloidal assembly in which attractions can be fine-tuned through salinity and, potentially, grafting density and temperature.

Cell-Free Gene Expression from DNA Brushes.

Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102-103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.

Boundary-Free Ribosome Compartmentalization by Gene Expression on a Surface.

The design of artificial cell models based on minimal surface-bound transcription–translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. Dense DNA brushes as localized sources of RNA and proteins serve as synthetic operons that have recently proven useful for the autonomous synthesis and assembly of cellular machines. Here, we studied ribosome compartmentalization in a minimal gene-expression reaction on a surface in contact with a macroscopic reservoir. We first observed the accumulation and colocalization of RNA polymerases, ribosomes, nascent RNAs and proteins, in dense DNA brushes using evanescent field fluorescence, showing transcription–translation coupling in the brush. Fluorescence recovery after photobleaching showed that ribosomes engaged in translation in the brush had a 4-fold slower diffusion constant. In addition, ribosomes in the brush had over a 10-fold higher local concentration relative to free ribosomes, creating a boundary-free functional ribosome-rich compartment. To decouple translation from transcription, we immobilized dense phases of ribosomes next to DNA brushes. We demonstrated that immobilized ribosomes were capable of protein synthesis, forming 2D subcompartments of active ribosome patterns induced and regulated by DNA brush layout of coding and inhibitory genes. Localizing additional molecular components on the surface will further compartmentalize gene-expression reactions.

Programming multi-protein assembly by gene-brush patterns and two-dimensional compartment geometry.

The assembly of protein machines in cells is precise, rapid, and coupled to protein synthesis with regulation in space and time. The assembly of natural and synthetic nanomachines could be similarly controlled by genetic programming outside the cell. Here, we present quasi-two-dimensional (2D) silicon compartments that enable programming of protein assembly lines by local synthesis from surface-immobilized DNA brushes. Using this platform, we studied the autonomous synthesis and assembly of a structural complex from a bacteriophage and a bacterial RNA-synthesizing machine. Local synthesis and surface capture of complexes provided high assembly yield and sensitive detection of spatially resolved assembly intermediates, with the 3D geometry of the compartment and the 2D pattern of brushes dictating the yield and mode of assembly steps. Localized synthesis of proteins in a single gene brush enhances their interactions, and displacement of their genes in separated brushes leads to step-by-step surface assembly. This methodology enables spatial regulation of protein synthesis, and deciphering, reconstruction and design of biological machine assembly lines.

Photo-printing of faceted DNA patchy particles

Patchy particles with shape complementarity can serve as building blocks for assembling colloidal superstructures. Alternatively, encoding information on patches using DNA can direct assembly into a variety of crystalline or other preprogrammed structures. Here, we present a tool where DNA is used both to engineer shape and to encode information on colloidal particles. Two reactive oil emulsions with different but complementary DNA (cDNA) brushes are assembled into CsCl-like crystalline lattices. The DNA brushes are recruited to and ultimately localized at the junctions between neighboring droplets, which gives rise to DNA-encoded faceted patches. The emulsions are then solidified by ultraviolet (UV) polymerization, producing faceted patchy particles. The facet size and DNA distribution are determined by the balance between the DNA binding energy and the elastic deformation energy of droplets. This method leads to a variety of new patchy particles with directional interactions in scalable quantities.

Cell-free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices.

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab-on-chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell-free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell-cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell-free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

DNA-Coated Microspheres and Their Colloidal Superstructures

Reversible and specific interaction between single-stranded DNA on colloidal particles have opened up a new path way of building up colloidal superstructures. DNA-coated microspheres can be bound with other particles with complementary DNA brushes below the melting temperature and can be unbound above the melting temperature. However, due to their random Brownian motion, the particles form random (or glassy) structures in most cases or small crystals when cooling is extremely slow. Therefore, toward programmed colloidal superstructures of DNA-coated microspheres, they should reconfigure their kinetically trapped random structure to equilibrium crystalline structures. While nanoparticles can be rearranged into a crystalline structure by a simple conformational change of relatively long DNA brush, microspheres with short DNA brushes cannot be rearranged only by a conformational change of brush. Instead, sub-diffusion of bound DNA-coated microspheres is necessary which can be possible only with uniform DNA coating with high areal density on microspheres. In this article, we have reviewed methods for the synthesis of high-density DNA-coated microspheres and their assembly into crystalline structures. We also discuss future research direction of DNA-coated microspheres.

Single-stranded DNA oligomer brush structure is dominated by intramolecular interactions mediated by the ion environment.

Single-stranded DNA (ssDNA) brushes, in which ssDNA oligomers are tethered to surfaces in dense monolayers, are being investigated for potential biosensing applications. The structure of the brush can affect the selectivity and the hybridization efficiency of the device. The structure is commonly thought to result from the balance of intramolecular interactions, intermolecular interactions within the monolayer, and molecule-surface interactions. Here, we test the hypothesis that ssDNA oligomer brush structure is dominated by intramolecular interactions. We use AFM to measure the height of an ssDNA brush and molecular dynamics to simulate the end-to-end distance, both as a function of ionic strength of the surrounding solution. The brush height and the molecule end-to-end distance match quantitatively, providing evidence that the brush structure is dominated by intramolecular interactions (mediated by ions). The physical basis of the intramolecular interactions is elucidated by the simulations.

Heterogeneous Morphology and Dynamics of Polyelectrolyte Brush Condensates in Trivalent Counterion Solution

Recent experiments have shown that trivalent ion, spermidine$^{3+}$, can provoke lateral microphase segregation in DNA brushes. Using molecular simulations and simple theoretical arguments, we explore the effects of trivalent counterions on polyelectrolyte brushes. At a proper range of grafting density, polymer size, and ion concentration, the brush polymers collapse heterogeneously into octopus-like surface micelles. Remarkably, the heterogeneity in brush morphology is maximized and the relaxation dynamics of chain and condensed ion are the slowest at the 1:3 stoichiometric concentration of trivalent ions to polyelectrolyte charge. A further increase of trivalent ion concentration conducive to a charge inversion elicits modest reswelling and homogenizes the morphology of brush condensate. Our study provides a new insight into the origin of the diversity in DNA organization in cell nuclei as well as the ion-dependent morphological variation in polyelectrolyte brush layer of biological membranes.

Effects of Chain-Chain Associations on Hybridization in DNA Brushes.

Hybridization of solution nucleic acids to DNA brushes is widely encountered in diagnostic and materials science applications. Typically, brush chain lengths of ten or more nucleotides are used to provide the needed sequence specificity and binding affinity. At these lengths, coincidental occurrence of complementary regions is expected to lead to associations between the nominally single-stranded brush chains due to intra- or interchain base pairing. This report investigates how these associations impact the brushes' hybridization activity toward complementary "target" sequences. Brushes were prepared from 20-mer chains with four-nucleotide-long "adhesive regions" through which neighboring chains could interact. The affinity and position of the adhesive region along the chain backbone were varied. DNA brushes were exposed to complementary solution targets, and the corresponding melting transitions were measured to estimate free energies of the brush-target hybridization. These results revealed that higher affinity adhesive regions more extensively suppressed brush hybridization relative to hybridization in solution. Associations near the middle of the chains were found to be more penalizing than those at the immobilized or the free end of the chains. Provided that the brush chains were close enough to associate, changes in brush density did not exert a significant effect on hybridization thermodynamics within the investigated coverage window. Comparison of the DNA brush results with those from commercial Affymetrix single-nucleotide-polymorphism (SNP) microarrays revealed agreement in the impact of chain associations on hybridization.

Transcription Driven Phase Separation in Chromatin Brush.

We theoretically predict the local density of nucleosomes on DNA brushes in a solution of molecules, which are necessary for transcription and the assembly of nucleosomes. Our theory predicts that in a confined space, DNA brushes show phase separation, where a region of relatively large nucleosomal occupancy coexists with a region of smaller nucleosomal occupancy. This phase separation is driven by an instability arising from the fact that the rate of transcription increases as the nucleosomal occupancy decreases due to the excluded volume interactions between nucleosomes and RNA polymerase during thermal diffusion and, in turn, nucleosomes are (in some cases) desorbed from DNA when RNA polymerase collides with nucleosomes during transcription. The miscibility phase diagram shows critical points, which are sensitive to the rate constants involved in transcription, the changes of interactions of DNA chain segments by assembling nucleosomes, and pressures that are applied to the brushes.

Transcription rates in DNA brushes.

We theoretically predict the rate of transcription (TX) in DNA brushes by introducing the concept of TX dipoles that takes into account the unidirectional motion of enzymes (RNAP) along DNA during transcription as correlated pairs of sources and sinks in the relevant diffusion equation. Our theory predicts that the TX rates dramatically change upon the inversion of the orientation of the TX dipoles relative to the substrate because TX dipoles modulate the concentrations of RNAP in the solution. Comparing our theory with experiments suggests that, in some cases, DNA chain segments are relatively uniformly distributed in the brush, in contrast to the parabolic profile expected for flexible polymer brushes.

Determination of Average Internucleotide Distance in Variable Density ssDNA Nanobrushes in the Presence of Different Cations Species

Controlling conformational and mechanical properties of single-stranded DNA (ssDNA) nanobrushes is crucial for the design of new, miniaturized DNA-based functional biosensors. In particular, counterions diffusion and binding affinity to DNA impact on ssDNAs curvature and flexibility and modify their binding properties. In order to highlight the role of cation electrostatic screening and molecular crowding on the conformational stability of DNA brushes, we propose here to use atomic force microscopy (AFM) and AFM-based lithography to create ssDNA assemblies of variable density and to analyze their collective response to changes of ionic strength. We confined ssDNA brushes with controlled surface densities within a biorepellent self-assembled monolayer. We then monitored the topographic brush height variation upon changing salt type (NaCl, KCl, CaCl2, and MgCl2) and concentration inside the liquid cell. We show that the measured height is related to scaling law of salt concentration, in agreement with the theory of polyelectrolyte brush. We find the same scaling exponent α = −1/6 for the different density regimes exploited. Using this scaling model to fit our experimental data, we quantified structural parameters such as the average internucleotide distance (d) for ssDNA brushes of different and estimated surface density (σ), featuring a strong dependence of d on different salts species.

Emergent properties of dense DNA phases toward artificial biosystems on a surface.

CONSPECTUS: The expression of genes in a cell in response to external signals or internal programs occurs within an environment that is compartmentalized and dense. Reconstituting gene expression in man-made systems is relevant for the basic understanding of gene regulation, as well as for the development of applications in bio- and nanotechnology. DNA polymer brushes assembled on a surface emulate a dense cellular environment. In a regime of significant chain overlap, the highly charged nature of DNA, its entropic degrees of freedom, and its interaction with transcription/translation machinery lead to emergent collective biophysical and biochemical properties, which are summarized in this Account. First, we describe a single-step photolithographic biochip on which biomolecules can be immobilized. Then, we present the assembly of localized DNA brushes, a few kilo-base pairs long, with spatially varying density, reaching a DNA concentration of ∼10(7) base pairs/μm(3), which is comparable to the value in E. coli. We then summarize the response of brush height to changes in density and mono- and divalent ionic strength. The balance between entropic elasticity and swelling forces leads to a rich phase behavior. At no added salt, polymers are completely stretched due to the osmotic pressure of ions, and at high salt they assume a relaxed coil conformation. Midrange, the brush height scales with ratio of density and ionic strength to the third power, in agreement with the general theory of polyelectrolyte brushes. In response to trivalent cations, DNA brushes collapse into macroscopic dendritic condensates with hysteresis, coexistence, and a hierarchy of condensation with brush density. We next present an investigation of RNA transcription in the DNA brush. In general, the brush density entropically excludes macromolecules, depleting RNA polymerase concentration in the brush compared to the bulk, therefore reducing transcription rate. The orientation of transcription promoters with respect to the surface also affects the rate with a lower value for outward compared to inward transcription, likely due to local changes of RNA polymerase concentrations. We hypothesize that equalizing the macromolecular osmotic pressure between bulk and brush with the addition of inert macromolecules would overcome the entropic exclusion of DNA associated proteins, and lead to enhanced biochemical activity. Finally, we present protein synthesis cascades in DNA brushes patterned at close proximity, as a step toward biochemical signaling between brushes. Examining the synthesis of proteins polymerizing into crystalline tubes suggests that on-chip molecular traps serve as nucleation sites for protein assembly, thereby opening possibilities for reconstituting nanoscale protein assembly pathways.

Dendritic and Nanowire Assemblies of Condensed DNA Polymer Brushes

We investigated the collective conformational response of DNA polymer brushes to condensation induced by the trivalent cation spermidine. DNA brushes, a few kilobase-pairs long, undergo a striking transition into macroscopic domains of collapsed chains with fractal dendritic morphology. Condensation is initiated by focal nucleation of a towerlike bundle, which laterally expands in a chain-reaction cascade of structural chain-to-chain collapse onto the surface. The transition exhibits the hallmarks of a first-order phase transition with grain boundaries, hysteresis, and coexistence between condensed and uncondensed phases. We found that an extended DNA conformation is maintained throughout the transition and is a prerequisite for the formation of large-scale dendritic domains. We identified a critical DNA density above which the nucleation propensity and growth rate sharply increase. We hypothesize that the ability of DNA-scaffolding proteins to modify the local DNA density within a genome may act as a dynamic and sensitive mechanism for spatial regulation of DNA transactions in vivo by selective condensation of chromosomal territories. By assembling a DNA brush along a patterned line narrower than twice the DNA contour length and tuning the local surface densities, we were able to initiate nucleation at a predefined location and induced growth of a single condensed nanowire over a distance 2 orders of magnitude longer than the single-chain contour. Our results demonstrate spatial control of condensation as a new tool for constructing DNA-based synthetic systems with important implications for regulation of DNA transactions on surfaces.

Network Formation by Cross-Hybridization of Complementary Strands to Grafted ssDNA.

When a low density brush of single-stranded DNA (ssDNA) targets end-grafted to a surface is immersed in a solution of complementary ssDNA probes, a regular brush of DNA duplexes is formed by 1:1 hybridization between probe and target DNA. We suggest that in higher density brushes of ssDNA this process competes with cross-hybridization of a target strand to several neighboring probe strands resulting in the formation of a cross-linked DNA network. We analyze a simple 2D model of a dense DNA brush and use analytic methods and computer simulations to find how the conditions for network formation depend on system size and DNA length. We argue that in 3D brushes cross-hybridization will nearly always lead to network formation and suggest that this may explain some intriguing results on dense DNA brushes. Experiments on DNA monolayers and concentrated DNA solutions that could test our predictions are proposed.

Spectroscopic Study of a DNA Brush Synthesized in Situ by Surface Initiated Enzymatic Polymerization

We used a combination of synchrotron-based X-ray photoelectron spectroscopy (XPS) and angle-resolved near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to study the chemical integrity, purity, and possible internal alignment of single-strand (ss) adenine deoxynucleotide (poly(A)) DNA brushes. The brushes were synthesized by surface-initiated enzymatic polymerization (SIEP) on a 25-mer of adenine self-assembled monolayer (SAM) on gold (A25-SH), wherein the terminal 3'-OH of the A25-SH serve as the initiation sites for SIEP of poly(A). XPS and NEXAFS spectra of poly(A) brushes were found to be almost identical to those of A25-SH initiator, with no unambiguous traces of contamination. Apart from the well-defined chemical integrity and contamination-free character, the brushes were found to have a high degree of orientational order, with an upright orientation of individual strands, despite their large thickness up to ~55 nm, that corresponds to a chain length of at least several hundred nucleotides for individual ssDNA molecules. The orientational order exhibited by these poly(A) DNA brushes, mediated presumably by base stacking, was found to be independent of the brush thickness as long as the packing density was high enough. The well-defined character and orientational ordering of the ssDNA brushes make them a potentially promising system for different applications.