DNA Rings Research Articles

A multiplexed circulating tumor DNA detection platform engineered from 3D-coded interlocked DNA rings

Circulating tumor DNA (ctDNA) is a critical biomarker not only important for the early detection of tumors but also invaluable for personalized treatments. Currently ctDNA detection relies on sequencing. Here, a platform termed three-dimensional-coded interlocked DNA rings (3D-coded ID rings) was created for multiplexed ctDNA identification. The ID rings provide a ctDNA recognition ring that is physically interlocked with a reporter ring. The specific binding of ctDNA to the recognition ring initiates target-responsive cutting via a restriction endonuclease; the cutting then triggers rolling circle amplification on the reporter ring. The signals are further integrated with internal 3D codes for multiplexed readouts. ctDNAs from non-invasive clinical specimens including plasma, feces, and urine were detected and validated at a sensitivity much higher than those obtained through sequencing. This 3D-coded ID ring platform can detect any multiple DNA fragments simultaneously without sequencing. We envision that our platform will facilitate the implementation of future personalized/precision medicine.

Programming ultrasensitive threshold response through chemomechanical instability

The ultrasensitive threshold response is ubiquitous in biochemical systems. In contrast, achieving ultrasensitivity in synthetic molecular structures in a controllable way is challenging. Here, we propose a chemomechanical approach inspired by Michell’s instability to realize it. A sudden reconfiguration of topologically constrained rings results when the torsional stress inside reaches a critical value. We use DNA origami to construct molecular rings and then DNA intercalators to induce torsional stress. Michell’s instability is achieved successfully when the critical concentration of intercalators is applied. Both the critical point and sensitivity of this ultrasensitive threshold reconfiguration can be controlled by rationally designing the cross-sectional shape and mechanical properties of DNA rings.

Equilibrium Conformation of Catenated DNA Networks in Slitlike Confinement.

A kinetoplast is a planar network of catenated DNA rings with topology that resembles that of chain mail armor. In this work, we use single-molecule experiments to probe the conformation of kinetoplasts confined to slits. We find that the in-plane size of kinetoplasts increases with degree of confinement, akin to the slitlike confinement of linear DNA. The change in kinetoplast size with channel height is consistent with the scaling prediction from a Flory-type approach for a 2D polymer. With an increase in extent of confinement, the kinetoplasts appear to unfold and take on more uniform circular shapes, in contrast to the broad range of conformations observed for kinetoplasts in bulk.

Dynamics and rheology of ring-linear blend semidilute solutions in extensional flow: Single molecule experiments

Ring polymers exhibit unique flow properties due to their closed chain topology. Despite recent progress, we have not yet achieved a full understanding of the nonequilibrium flow behavior of rings in nondilute solutions where intermolecular interactions greatly influence chain dynamics. In this work, we directly observe the dynamics of DNA rings in semidilute ring-linear polymer blends using single molecule techniques. We systematically investigate ring polymer relaxation dynamics from high extension and transient and steady-state stretching dynamics in a planar extensional flow for a series of ring-linear blends with varying ring fraction. Our results show multiple molecular subpopulations for ring relaxation in ring-linear blends, as well as large conformational fluctuations for rings in a steady extensional flow, even long after the initial transient stretching process has subsided. We further quantify the magnitude and characteristic time scales of ring conformational fluctuations as a function of blend composition. Interestingly, we find that the magnitude of ring conformational fluctuations follows a nonmonotonic response with increasing ring fraction, first increasing at low ring fraction and then substantially decreasing at large ring fraction in ring-linear blends. A unique set of ring polymer conformations are observed during the transient stretching process, which highlights the prevalence of molecular individualism and supports the notion of complex intermolecular interactions in ring-linear polymer blends. In particular, our results suggest that transient intermolecular structures form in ring-linear blends due to a combination of direct forces due to linear chains threading through open rings and indirect forces due to hydrodynamic interactions; these combined effects lead to large conformational fluctuations of rings over distributed time scales. Taken together, our results provide a new molecular understanding of ring polymer dynamics in ring-linear blends in the nonequilibrium flow.

Editors' Note: Novel ALDH5A1 Variants and Genotype: Phenotype Correlation in SSADH Deficiency

<h3>Abstract</h3> Knots and supercoiling are both introduced in bacterial plasmids by catalytic processes involving DNA strand passages. While the effects on plasmid organization has been extensively studied for knotting and supercoiling taken separately, much less is known about their concurrent action. Here, we use molecular dynamics simulations and oxDNA, an accurate mesoscopic DNA model, to study the kinetic and metric changes introduced by complex (five-crossing) knots and supercoiling in 2kbp-long DNA rings. We find several unexpected results. First, the conformational ensemble is dominated by two distinct states, differing in branchedness and knot size. Secondly, fluctuations between these states are as fast as the metric relaxation of unknotted rings. In spite of this, certain boundaries of knotted and plectonemically-wound regions can persist over much longer timescales. These pinned regions involve multiple strands that are interlocked by the cooperative action of topological and supercoiling constraints. Their long-lived character may be relevant for the simplifying action of topoisomerases.

Front Cover: Stepwise Strategy for One‐Pot Synthesis of Single‐Stranded DNA Rings from Multiple Short Fragments (6/2021)

Front Cover: Stepwise Strategy for One‐Pot Synthesis of Single‐Stranded DNA Rings from Multiple Short Fragments (6/2021)

Programming CircLigase Catalysis for DNA Rings and Topologies.

Circular single-stranded (ss) DNA is an essential element in rolling circle amplification and many DNA nanotechnology constructions. It is commonly synthesized from linear ssDNA by a ligase, which nevertheless suffers from low and inconsistent efficiency due to the simultaneous formation of concatemeric byproducts. Here, we design an intramolecular terminal hybridization strategy to program the ring formation catalytic process of CircLigase, a thermostable RNA ligase 1 that can ligate ssDNA in an intramolecular fashion. With the enthalpy gained from the programmed hybridization to override disfavored entropic factors associated with end coupling, we broke the limit of natural CircLigase on circularization of ssDNA, realizing over 75% yields of byproduct-free monomeric rings on a series of hundred-to-half-kilo-based linear DNAs. We found that this hybridization strategy can be twisted from intra- to intermolecular to also program CircLigase to efficiently and predominantly join one ssDNA strand to another. We focused on DNA rings premade by CircLigase and demonstrated their utility in elevating the preparation, quantity, and quality of DNA topologies. We expect that the new insights on engineering CircLigase will further promote the development of nucleic acid biotechnology and nanotechnology.

Stepwise Strategy for One-Pot Synthesis of Single-Stranded DNA Rings from Multiple Short Fragments.

Cyclic rings of single-stranded (ss) DNA have various unique properties, but wider applications have been hampered by their poor availability. This paper reports a convenient one-pot method in which these rings are efficiently synthesized by using T4 DNA ligase through convergent cyclization of easily available short DNA fragments. The key to the present method is to separate all the splint oligonucleotides into several sets, and add each set sequentially at an appropriate interval to the solutions containing all the short DNA fragments. Compared with simple one-pot strategies involving simultaneous addition of all the splints at the beginning of the reaction, both the selectivity and the yields of target ssDNA rings are greatly improved. This convergent method is especially useful for preparing large-sized rings that are otherwise hard to obtain. By starting from six short DNA fragments (71-82 nt), prepared by a DNA synthesizer, a ssDNA ring of 452-nt size was synthesized in 35 mol % yield and in high selectivity. Satisfactorily pure DNA rings were obtainable simply by treating the crude products with exonuclease.

An analytical method to connect open curves for modeling protein-bound DNA minicircles

We introduce an analytical method to generate the pathway of a closed protein-bound DNA minicircle. We develop an analytical equation to connect two open curves smoothly and use the derived expressions to join the ends of two helical pathways and form models of nucleosome-decorated DNA minicircles. We find that the simplest smooth connector which satisfies the boundary conditions at the end points and the length requirement for such connections to be a quartic function on the xy-plane and linear along the z-direction. This is a general method which can be used to connect any two open curves with well defined mathematical definitions as well as pairs of discrete systems found experimentally. We used this method to describe the configurations of torsionally relaxed, 360-base pair DNA rings with two evenly-spaced, ideal nucleosomes. We considered superhelical nucleosomal pathways with different levels of DNA wrapping and allowed for different inter-nucleosome orientations. We completed the DNA circles with the smooth connectors and studied the associated bending and electrostatic energies for different configurations in the absence and presence of salt. The predicted stable states bear close resemblance to reconstituted minicircles observed under low and high salt conditions.

Ionic Effects on the Equilibrium Conformation of Catenated DNA Networks

In this work, we study the ionic effects on the equilibrium size and shape of kinetoplasts, a two-dimensional (2D) network of catenated DNA rings. With increasing ionic strength from 5 to 200 mM, we observe a decrease in kinetoplast size, primarily driven by the long-range electrostatic interactions that give rise to a change in effective DNA width. A fit of the experimentally measured kinetoplast size versus effective width yields a scaling exponent of 0.38. To probe the quantitative effects of ionic strength on kinetoplast size, we develop a scaling argument based on a generalized Flory approach for a 2D polymer represented as monomers on an open lattice. Interestingly, while ionic strength has a significant effect on kinetoplast size, we find that it does not impact the kinetoplast shape.

Robust Storage of Chinese Language in a Pool of Small Single-Stranded DNA Rings and Its Facile Reading-Out

Abstract Archival storage in DNA is one of the most challenging themes in rapidly growing information technology. In addition, its practical applications are more difficult due to complicated data analysis, instability of long and linear DNA strands (&amp;gt;1000 nt), and other factors. In the present study, we have developed a simple and eminent DNA-based storage system in which small DNA rings are employed as memory units. Compared with previous approaches, this methodology has advantages of robustness, low cost, convenience and so on. In high density, Chinese language was directly stored in a pool of 76-nt-long single-stranded DNA rings (designated as “Info-Store”), in which each ring memorized the index number and five Chinese characters (or marks). During “Read-Out”, all the ssDNA rings in the pool were simultaneously amplified by hyper-branched rolling cycle amplification (HRCA), and their sequences were accurately determined by a portable MinION sequencer aided by a personal computer. Then, the original Chinese text was precisely and smoothly decoded by simple data analysis.

An orbitron-like 3D DNA clip-based nanomachine and its application for sensitive fluorescent bioassay of MicroRNA

Herein, an orbitron-like three-dimensional (3D) DNA clip-based nanomachine was proposed for ultrasensitive fluorescent bioassay of microRNA, which was constructed by mechanically interlocking double-DNA-ring with two single-stranded DNAs, performing an orbitron-like 3D structure with double freely rotated DNA rings as the open state. In the presence of target microRNA, the proposed orbitron-like 3D DNA clip can alter its structure from open to closed state in identification of the target microRNA, generating the closure between the previously modified fluorescent dyes and the quenchers to perform a “signal off” fluorescent signal correlated with the concentration of target microRNA. Compared with the normal DNA nanomachines, such as DNA tweezers constructed by self-assembly of three single-stranded DNAs which regulated the open and closed states on the basis of linear conformational changes, the proposed 3D DNA clip-based nanomachine with high mechanical rigidity realized the conformational changes in 3D space with the assistance of target microRNA, which could effectively increase the adjustable distance range and reduce the background signal. Furthermore, the 3D DNA clip-based nanomachine was applied in the fluorescent detection of microRNA-21 with favorable performances for the sensitive detection of microRNA in cells, providing a new avenue for early clinical diagnoses of disease.

Fluorescent aptasensors for parallel analysis of biomolecules based on interlocked DNA catenane nanomachines

Stimuli-responsive DNA catenane nanomachines have received considerable interest in the area of DNA nanotechnology. However, the sensing and bio-sensing applications of DNA catenane nanomachines are rarely explored. Herein, the biological small molecule and protein responsive DNA catenane nanomachines were designed by utilizing the specific aptamer/target interaction. In the presence of the target, the blocked catalytically inactive DNA catenane can be activated, while generating the metal ion-dependent DNAzyme as the signal output unit. The method allows the fluorescent detection of ATP and lysozyme with a detection limit of 20 nM and 200 pM, respectively. More importantly, through the use of different fluorophores as labels, the parallel analysis of ATP and lysozyme in the mixed solution was demonstrated based on two interlocked DNA catenanes. Compared with linear scaffold, the interlocked DNA rings showed enhanced stability against enzyme degradation and easily realized intramolecular reconfiguration. Due to its inherent advantages, the interlocked DNA catenane holds great promise in the field of DNA based sensors and nanodevices.

Growth induced buckling of morphoelastic rod in viscous medium**Project supported by the National Natural Science Foundation of China (Grant Nos. 11772141 and 11262019) and the State Scholarship Fund of China Scholarship Council (Grant No. 201708370030).

Biological growth is a common phenomenon in nature, and some organisms such as DNA molecules and bacterial filaments grow in viscous media. The growth induced instability of morphoelastic rod in a viscous medium is studied in this paper. Based on the Kirchhoff kinetic analogy method, the mechanical model for growing elastic thin rod in the viscous medium is established. A perturbation analysis is used to analyze the stability of the growing elastic rod in the viscous medium. We apply the results into planar growing ring and get its criterion of instability. Take the criterion into DNA ring to discuss the influence of viscous resistance on its instability.

Adjusting Linking Strands to Form Size-Controllable DNA Origami Rings.

DNA origami is a powerful tool in nanotechnology that can be used to construct arbitrary structures for several nanoengineering applications. Generally, the more complex and sophisticated the construction, the greater is the number of origamis and connection strands that will be needed. Therefore, developing an effective and low-cost method for multiform DNA architecture is important in nanoengineering. Here, we adopted an oblique linking strategy to connect cross-shaped DNA origami with a controlled curing angle. The size of the DNA rings ranged from four blocks of approximately 200 nm to eleven blocks of c.a. 600 nm. We observed that the minimum size of the DNA ring structure was limited by the width of a single block. The largest rings were negatively affected by thermodynamic randomness, and thus, DNA rings consisting of more than eleven blocks were not observed. This strategy facilitates the generation of various DNA origami rings, whose size can be controlled by adjusting the length of the connection strands.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

TacoxDNA: A user-friendly web server for simulations of complex DNA structures, from single strands to origami.

Simulations of nucleic acids at different levels of structural details are increasingly used to complement and interpret experiments in different fields, from biophysics to medicine and materials science. However, the various structural models currently available for DNA and RNA and their accompanying suites of computational tools can be very rarely used in a synergistic fashion. The tacoxDNA webserver and standalone software package presented here are a step toward a long-sought interoperability of nucleic acids models. The webserver offers a simple interface for converting various common input formats of DNA structures and setting up molecular dynamics (MD) simulations. Users can, for instance, design DNA rings with different topologies, such as knots, with and without supercoiling, by simply providing an XYZ coordinate file of the DNA centre-line. More complex DNA geometries, as designable in the cadnano, CanDo and Tiamat tools, can also be converted to all-atom or oxDNA representations, which can then be used to run MD simulations. Though the latter are currently geared toward the native and LAMMPS oxDNA representations, the open-source package is designed to be further expandable. TacoxDNA is available at http://tacoxdna.sissa.it. © 2019 Wiley Periodicals, Inc.

Hairpin/DNA ring ternary probes for highly sensitive detection and selective discrimination of microRNA among family members

The detection and quantification of microRNA (miRNA) plays essential roles in clinical and biomedical research. Yet, it is of major challenge to sense miRNA with high degree of selectivity and sensitivity due to its unique characteristics of short length, similarity of sequence among family members and low abundance. Here, with the design of a new hairpin/DNA ring ternary probe, we describe the development of a rolling circle amplification (RCA) method for sensitively and selectively sensing miRNA from cancer cells. The target miRNA binds the hairpin/DNA ring probes through toehold-mediated strand displacement (TSD) to form the ternary structures, in which the bound miRNA and DNA ring are respectively used as the primer and template to realize RCA, leading to the generation of many repeated metal ion-dependent DNAzyme sequences. The fluorescently quenched hairpin signal probes can be cyclically cleaved by these DNAzyme sequences with co-existence of the corresponding metal ions in buffer to show drastically enhanced fluorescence recovery for highly sensitive sensing of miRNA in the range between 10 fM and 10 nM with a detection limit of 1.51 fM. Besides, owing to the high base variation discrimination ability of TSD, selective detection of the target miRNA among the corresponding family members can be achieved by this method. Moreover, such a method can also be employed to differentiate miRNA expression variations in cancer cells for screening potential therapeutic drugs.

Topologically Linked Chains in Confinement.

We examine how channel confinement affects the equilibrium properties of topologically linked ring polymers and, by contrast, of equivalent unlinked rings, too. By performing extensive simulations of semiflexible rings of different chain length, N, and channel diameter, D, we discover three notable properties purely due to linking. First, upon entering the weak confinement regime, the length of the physically linked portion, The, becomes independent of chain length. Next, even when confinement is strong enough to pull apart and segregate unlinked rings, stays much larger than in the highly stretched limit. Finally, at fixed N, varies approximately as D0.5, and we provide a simple scaling argument for this power-law behavior. These properties, which mayhold for different link topologies, can be tested by current experimental setups on DNA rings confined in microchannels. Moreover, they could be relevant for the efficient in vivo unlinking of newly replicated bacterial chromosomes.

Calculation of \u03c0 and Classification of Self-avoiding Lattices via DNA Configuration

Numerical simulation (e.g. Monte Carlo simulation) is an efficient computational algorithm establishing an integral part in science to understand complex physical and biological phenomena related with stochastic problems. Aside from the typical numerical simulation applications, studies calculating numerical constants in mathematics, and estimation of growth behavior via a non-conventional self-assembly in connection with DNA nanotechnology, open a novel perspective to DNA related to computational physics. Here, a method to calculate the numerical value of π, and way to evaluate possible paths of self-avoiding walk with the aid of Monte Carlo simulation, are addressed. Additionally, experimentally obtained variation of the π as functions of DNA concentration and the total number of trials, and the behaviour of self-avoiding random DNA lattice growth evaluated through number of growth steps, are discussed. From observing experimental calculations of π (πexp) obtained by double crossover DNA lattices and DNA rings, fluctuation of πexp tends to decrease as either DNA concentration or the number of trials increases. Based upon experimental data of self-avoiding random lattices grown by the three-point star DNA motifs, various lattice configurations are examined and analyzed. This new kind of study inculcates a novel perspective for DNA nanostructures related to computational physics and provides clues to solve analytically intractable problems.