DNA Rings Research Articles

Target-Triggered Multiple-Polarity-Switchable Multiplexed Photoelectrochemical Platform.

Convenient and accurate quantification of disease-relevant multitargets is essential for community disease screening. However, in the field of photoelectrochemical (PEC) sensors for multisubstance detection, research on the continuous detection of multiple targets using a polarity-switching mode is scarce. In this study, a multiplexed PEC bioassay was developed based on a target-triggered "anodic-cathodic-anodic" multiple-polarity-switchable mode. Employing miRNA-21 and miRNA-141 as model analytes, the photosensitive material combinations of Cu2O/gold nanoparticles (AuNPs)/TiO2 and CdS/AuNPs/TiO2 were successively formed through the specific binding of different whisker branches of Whisker-DNA to Cu2O-H1 and the CdS-tripod DNA ring, respectively. This process reverses the photocurrent polarity from anodic to cathodic and then back to anodic upon detecting different targets, resulting in the high-sensitivity quantification of various biological targets with reduced interference. To enhance the device's utility and affordability in community disease screening, integrating a capacitor and a multimeter-smartphone connection simplifies the assembly and reduces costs. In developing the PEC sensor, the device demonstrated linear detection ranges for miRNA-21 and miRNA-141 from 0.01 fM to 10 nM. Detection limits for miRNA-21 and miRNA-141 were established at 3.2 and 4.3 aM, respectively. The innovative target-triggered multiple-polarity-switchable mode offers adaptability for other multitarget detections by simply modifying the structure of the whisker branches and the combination of photosensitive materials.

Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick.

The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2-4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5-bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are also helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.

Triggered contraction of self-assembled micron-scale DNA nanotube rings

Contractile rings are formed from cytoskeletal filaments during cell division. Ring formation is induced by specific crosslinkers, while contraction is typically associated with motor protein activity. Here, we engineer DNA nanotubes and peptide-functionalized starPEG constructs as synthetic crosslinkers to mimic this process. The crosslinker induces bundling of ten to hundred DNA nanotubes into closed micron-scale rings in a one-pot self-assembly process yielding several thousand rings per microliter. Molecular dynamics simulations reproduce the detailed architectural properties of the DNA rings observed in electron microscopy. Theory and simulations predict DNA ring contraction – without motor proteins – providing mechanistic insights into the parameter space relevant for efficient nanotube sliding. In agreement between simulation and experiment, we obtain ring contraction to less than half of the initial ring diameter. DNA-based contractile rings hold promise for an artificial division machinery or contractile muscle-like materials.

Bacterial nucleoid is a riddle wrapped in a mystery inside an enigma.

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery-facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design-even though its main intracellular transportation mode is free diffusion.

Olympic gels formed through catenation of dsDNA rings regulated by topoisomerase II: A coarse-grained model.

Topological regulation of DNA by topoisomerases in cells is very crucial for life. We propose a coarse-grained model to study the catenation process of double-stranded DNA (dsDNA) rings regulated by topoisomerase II (TOP2) and provide a computational method to characterize the topological structures of the Olympic gels obtained. The function of TOP2 in the catenation of dsDNA rings is implicitly fulfilled by operating the length of a stretchable catch bond in the dsDNA ring. After the catenation reaction of initially noncatenated dsDNA rings in the solution, the Olympic gel is obtained and the interlocked topology of the dsDNA rings can be characterized by a computational method derived from the HOMFLY polynomial, based on which the catenation degree and the complexity of catenation are quantified. Detailed dependence of the catenation degree and the complexity of the catenated topology on key parameters, including the size of the transient broken gap and the duration time of the break on the dsDNA ring during operation by TOP2, the initial molar ratio of TOP2 to the dsDNA rings, and the reaction temperature, has been investigated.

Enzymatic cleaving of entangled DNA rings drives scale-dependent rheological trajectories.

DNA, which naturally occurs in linear, ring, and supercoiled topologies, frequently undergoes enzyme-driven topological conversion and fragmentation in vivo, enabling it to perform a variety of functions within the cell. In vitro, highly concentrated DNA polymers form entanglements that yield viscoelastic properties dependent on the topologies and lengths of the DNA. Enzyme-driven alterations of DNA size and shape therefore offer a means of designing active materials with programmable viscoelastic properties. Here, we incorporate multi-site restriction endonucleases into dense DNA solutions to linearize and fragment circular DNA molecules. We pair optical tweezers microrheology with differential dynamic microscopy and single-molecule tracking to measure the linear and nonlinear viscoelastic response and transport properties of entangled DNA solutions over a wide range of spatiotemporal scales throughout the course of enzymatic digestion. We show that, at short timescales, relative to the relaxation timescales of the polymers, digestion of these 'topologically-active' fluids initially causes an increase in elasticity and relaxation times followed by a gradual decrease. Conversely, for long timescales, linear viscoelastic moduli exhibit signatures of increasing elasticity. DNA diffusion, likewise, becomes increasingly slowed, in direct opposition to the short-time behavior. We hypothesize that this scale-dependent rheology arises from the population of small DNA fragments, which increases as digestion proceeds, driving self-association of larger fragments via depletion interactions, giving rise to slow relaxation modes of clusters of entangled chains, interspersed among shorter unentangled fragments. While these slow modes likely dominate at long times, they are presumably frozen out in the short-time limit, which instead probes the faster relaxation modes of the unentangled population.

Cooperative Rheological State-Switching of Enzymatically-Driven Composites of Circular DNA And Dextran.

Polymer topology, which plays a principal role in the rheology of polymeric fluids, and non-equilibrium materials, which exhibit time-varying rheological properties, are topics of intense investigation. Here, composites of circular DNA and dextran are pushed out-of-equilibrium via enzymatic digestion of DNA rings to linear fragments. These time-resolved rheology measurements reveal discrete state-switching, with composites undergoing abrupt transitions between dissipative and elastic-like states. The gating time and lifetime of the elastic-like states, and the magnitude and sharpness of the transitions, are surprisingly decorrelated from digestion rates and non-monotonically depend on the DNA fraction. These results are modeled using sigmoidal two-state functions to show that bulk state-switching can arise from continuous molecular-level activity due to the necessity for cooperative percolation of entanglements to support macroscopic stresses. This platform, coupling the tunability of topological composites with the power of enzymatic reactions, may be leveraged for diverse material applications from wound-healing to self-repairing infrastructure.

Tuning the topology of a two-dimensional catenated DNA network

Molecular topology of polymers plays a key role in determining their physical properties. We studied herein the topological effects on the static and dynamic properties of a 2D catenated network of DNA rings called a kinetoplast. Restriction enzymes that cleave DNA at sequence-specific sites are used to selectively cut and remove rings from the network and hence tune the molecular topology while maintaining overall structural integrity. We find that topology has minimal effects over the spatial extension of the 2D network; however, it significantly affects the relaxation behavior. The shape fluctuations of the network are governed by two distinct characteristic time scales attributed to the thermal fluctuations and confinement of the network. The relationship between the time constant of thermal relaxation and the amplitude of anisotropy fluctuations yields a universal scaling. Interestingly, this scaling is independent of the detailed arrangements of rings and/or perforation within the catenated networks. This study provides a route to tune the elastic properties of 2D catenated DNA networks and other polymeric materials by modifying the underlying topology in a rational and highly controllable manner.

Dynamic concatenation of ring DNA leads to non-equilibrium viscoelasticity tuned by DNA size and concentration.

Topoisomerases can cleave, twist, untwist and reconnect ring DNA to enable diverse biological processes including DNA replication and repair, as well as the formation of concatenated structures such as kinetoplasts. At the same time, dense solutions of topologically-varying DNA have been shown to exhibit unique viscoelastic properties that can be tuned by DNA concentration, size and topology. Here, we use dynamic light scattering and particle-tracking microrheology to characterize the non-equilibrium viscoelastic properties of dense solutions of ring DNA undergoing dynamic concatenation via the linking and unlinking action of Topoisomerase II. We show that the frequency-dependent viscoelastic properties of these active Olympic ring hydrogels can be precisely tuned by varying the size and concentration of the DNA rings as well as the rate at which Topoisomerase II links and unlinks the DNA. Our work sheds new light on the oft overlooked role of varying viscoelasticity on biological processes that alter DNA topology, including the formation and restructuring of kinetoplasts, and the unwinding of DNA for replication and repair. Future work will build on this bio-inspired platform by incorporating additional enzymes that fragment and ligate DNA for in situ alteration of DNA length.

Dynamic concatenation leads to anomalous transport of ring DNA in concentrated solutions.

Circular DNA polymers have been widely studied due to their biological relevance as well as the unique anomalous transport properties they exhibit in concentrated solutions and blends with linear DNA. In cells, topoisomerase II relaxes torsional strain in circular DNA by cutting and then reconnecting one of the strands of the double-stranded polymer, enabling replication, transcription, and repair. When topoisomerase II acts on nicked circular DNA at high concentration, the relaxed rings can concatenate, forming Olympic ring structures reminiscent of kinetoplasts. The rate of topoisomerase activity as well as the concentration and size of the DNA can tune the nonequilibrium dynamics and conformations of the concatemers. Here, we visualize the fluorescent-labeled DNA rings comprising the concatemers and use particle tracking and differential dynamic microscopy to characterize the dynamics and time-varying size and shape of the actively linking and unlinking molecules. Beyond the insight our work provides to biological processes mediated by active restructuring of DNA, the complex fluids we engineer and study may have applications in the design of biocompatible active materials for cellular repair, toxin filtration and drug delivery.

Functionalized DNA-Origami-Protein Nanopores Generate Large Transmembrane Channels with Programmable Size-Selectivity.

The DNA-origami technique has enabled the engineering of transmembrane nanopores with programmable size and functionality, showing promise in building biosensors and synthetic cells. However, it remains challenging to build large (>10 nm), functionalizable nanopores that spontaneously perforate lipid membranes. Here, we take advantage of pneumolysin (PLY), a bacterial toxin that potently forms wide ring-like channels on cell membranes, to construct hybrid DNA-protein nanopores. This PLY-DNA-origami complex, in which a DNA-origami ring corrals up to 48 copies of PLY, targets the cholesterol-rich membranes of liposomes and red blood cells, readily forming uniformly sized pores with an average inner diameter of ∼22 nm. Such hybrid nanopores facilitate the exchange of macromolecules between perforated liposomes and their environment, with the exchange rate negatively correlating with the macromolecule size (diameters of gyration: 8-22 nm). Additionally, the DNA ring can be decorated with intrinsically disordered nucleoporins to further restrict the diffusion of traversing molecules, highlighting the programmability of the hybrid nanopores. PLY-DNA pores provide an enabling biophysical tool for studying the cross-membrane translocation of ultralarge molecules and open new opportunities for analytical chemistry, synthetic biology, and nanomedicine.

Topology-driven spatial organization of ring polymers under confinement.

Entropic repulsion between DNA ring polymers under confinement is a key mechanism governing the spatial segregation of bacterial DNA before cell division. Here we establish that "internal" loops within a modified-ring polymer architecture enhance entropic repulsion between two overlapping polymers confined in a cylinder. Interestingly, they also induce entropy-driven spatial organization of polymer segments as seen in vivo. Here we design polymers of different architectures in our simulations by introducing a minimal number of cross-links between specific monomers along the ring-polymer contour. The cross-links are likely induced by various bridging proteins inside living cells. We investigate the segregation of two polymers with modified topologies confined in a cylinder, which initially had spatially overlapping configurations. This helps us to identify the architectures that lead to higher success rates of segregation. We also establish the mechanism that leads to localization of specific polymer segments. We use the blob model to provide a theoretical understanding of why certain architectures lead to enhanced entropic repulsive forces between the polymers. Lastly, we establish a correspondence between the organizational patterns of the chromosome of the C.crescentus bacterium and our results for a specifically designed polymer architecture. However, the principles outlined here pertaining to the organization of polymeric segments are applicable to both synthetic and biological polymers.

Nanorings to Probe Mechanical Stress of Single-Stranded DNA Mediated by the DNA Duplex.

The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins.

The Arabidopsis APOLO and human UPAT sequence-unrelated long noncoding RNAs can modulate DNA and histone methylation machineries in plants

BackgroundRNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity.ResultsHere, we show that in addition to the PRC1-component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrate that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we show that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter and globally alter the Arabidopsis transcriptome in a similar manner.ConclusionsCollectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation through the interaction with VIM1, and indicates that evolutionary unrelated lncRNAs with potentially conserved structures may exert similar functions by interacting with homolog partners.

Developmental Self-Assembly of a DNA Ring with Stimulus-Responsive Size and Growth Direction.

Developmental self-assembly of DNA nanostructures provides an ideal platform for studying the power and programmability of kinetically controlled structural growth in engineered molecular systems. Triggered initiation and designated sequencing of assembly and disassembly steps have been demonstrated in structures with branches and loops. Here we introduce a new strategy for selectively activating distinct subroutines in a developmental self-assembly program, allowing structures with distinct properties to be created in response to various molecular signals. We demonstrate this strategy in triggered self-assembly of a DNA ring, the size and growth direction of which are responsive to a key molecule. We articulate that reversible assembly steps with slow kinetics at appropriate locations in a reaction pathway could enable multiple populations of structures with stimulus-responsive properties to be simultaneously created in one developmental program. These results open up a broad design space for the self-assembly of molecules with adaptive behaviors toward advanced control in synthetic materials and molecular motors.

Casimersen for the treatment of Duchenne muscular dystrophy

Casimersen is a single-stranded, antisense phosphorodiamidate morpholino oligonucleotide where six-membered morpholino rings substitute the ribofuranose rings of the natural DNA and RNA backbone. The morpholino is conjugated to the following monomer via a charge-neutral phosphorodiamidate instead of the phosphodiester linkage containing a negative charge. Phosphorodiamidate morpholino oligonucleotides are resistant to enzymatic degradation and their lack of charge facilitates cellular penetration.

Quantitative determination of circular DNA fragments of the T- and B-cell receptor TREC/KREC in patients with ataxia-telangiectasia with a mutation in the ATM gene

Purpose. Evaluate the diagnostic significance of quantitative determination of T- and B-cell receptor DNA ring fragments in peripheral blood of patients for diagnosis of ataxia-teleangiectasia. Materials and methods. The subjects of the study were peripheral blood samples from 14 patients diagnosed with ataxia-teleangiectasia aged 4.9 (1.6; 14.4) years with mutations in the ATM gene and from 40 healthy children aged 11.0 (6.7; 14.8) years. Results and discussion. Patients with ataxia-teleangiectasia revealed a significant decrease in TREC and KREC values (p<0.05) compared to the control group of healthy children. Diagnostic sensitivity and specificity concerning the use of TREC determination (92.8% and 93.4% respectively) and KREC (85.7% and 96.7% respectively) in patients with ataxia-telangioectasia allow the use of this analysis in laboratory practice. Conclusion. The method of quantitative determination of TREC and KREC due to high values of sensitivity and specificity of the applied technique can be used in the diagnosis of ataxia-teleangiectasia.

Tumor-targeting [2]catenane-based grid-patterned periodic DNA monolayer array for in vivo theranostic application.

DNA nanotechnology is often used to build various nano-structures for signaling and/or drug delivery, but it essentially suffers from several major limitations, such as a large number of DNA strands and limited targeting ligands. Moreover, there is no report on in vivo two-dimensional DNA arrays because of various technical challenges. By cross-catenating two palindromic DNA rings, herein, we demonstrate a catenane-based grid-patterned periodic DNA monolayer array ([2]GDA) capable of preferentially accumulating in tumor tissues without any targeting ligands, with a thickness equal to the double-helical DNA monolayer (nearly 2 nm). The structural flexibility of [2]GDA enabled it to fold into a spherical object in solution, favoring cellular uptake. Thus, its cellular internalization activity was comparable with that of the commercial lipofectamine 3000. Moreover, [2]GDA retained the structural integrity over 24 h incubation in biological solutions, achieving a 360-fold improvement in in vivo stability. Significantly, anticancer drug-loaded [2]GDA exhibits desirable therapeutic efficacy in tumor-bearing animals without detectable side effects.

Genome-Wide DNA Methylation Protocol for Epigenetics Studies.

Epigenetic modification are heritable changes in gene expression not encoded by the DNA sequence therefore playing a significant role in a broad range of biological processes and diseases.A key player of the epigenetic modifications is the DNA methylation, a process involving the covalent transfer of a methyl group to the C-5 position of the cytosine ring of DNA forming 5-methylcytosine (5mC), catalyzed by DNA methyltransferases. Altering the structure of the chromatin, DNA methylation has the potential to down-regulate gene expression.The here presented protocol shows a method to obtain DNA samples ready for NGS sequencing for genome-wide methylation analysis.

Computer simulations of melts of ring polymers with nonconserved topology: A dynamic Monte Carlo lattice model.

We present computer simulations of a dynamic Monte Carlo algorithm for polymer chains on a fcc lattice which explicitly takes into account the possibility to overcome topological constraints by controlling the rate at which nearby polymer strands may cross through each other. By applying the method to systems of interacting ring polymers at melt conditions, we characterize their structure and dynamics by measuring, in particular, the amounts of knots and links which are formed during the relaxation process. In comparison with standard melts of unknotted and unconcatenated rings, our simulations demonstrate that the mechanism of strand crossing makes polymer dynamics faster provided the characteristic timescale of the process is smaller than the typical timescale for chain relaxation in the unperturbed state, in agreement with recent experiments employing solutions of DNA rings in the presence of the type II topoisomerase enzyme. In the opposite case of slow rates the melt is shown to become slower, and this prediction may be easily validated experimentally.