Hybridization Kinetics Research Articles

Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA.

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

Efficient Simulation of Arbitrary Multicomponent First-Order Binding Kinetics for Improved Assay Design and Molecular Assembly.

Traditional enzyme-linked immunosorbent assay (ELISA), long the workhorse for specific target protein detection using microplate wells, is nearing its fundamental limit of sensitivity. New opportunities in health care call for in vitro diagnostic tests with ultrahigh sensitivity. Magnetic bead-based sandwich immunoassay formats have been developed that can reach unprecedented sensitivities, orders of magnitude better than are allowed for by the rate constants for a single ligand–receptor interaction. However, these ultrahigh sensitivity assays are vulnerable to a host of confounding factors, including nonspecific binding from background molecules and loss of low-abundance target to tube walls and during wash steps. Moreover, the optimization of workflow is often time-consuming and expensive. In this work, we present a simulation tool that allows users to graphically define arbitrary binding assays, including fully reversible first-order binding kinetics, timed addition of extra components, and timed wash steps. The tool is freely available as a user-friendly webapp. The framework is lightweight and fast, allowing for inexpensive simulation and visualization of arbitrarily complex assay schemes, including but not limited to digital immunoassays, DNA hybridization, and enzyme kinetics, for validation and optimization of assay designs without requiring any programming knowledge from the user. We demonstrate some of these capabilities and provide practical guidance on assay simulation design.

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

The physical–chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal’s amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M−1·s−1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

Quantification of Strand Accessibility in Biostable DNA Origami with Single-Staple Resolution.

DNA-based nanostructures are actively gaining interest as tools for biomedical and therapeutic applications following the recent development of protective coating strategies prolonging structural integrity in physiological conditions. For tailored biological action, these nanostructures are often functionalized with targeting or imaging labels using DNA base pairing. Only if these labels are accessible on the structure’s surface will they be able to interact with their intended biological target. However, the accessibility of functional sites for different geometries and environments, specifically after the application of a protective coating, is currently not known. Here, we assay this accessibility on the level of single handle strands with two- and three-dimensional resolution using DNA-PAINT and show that the hybridization kinetics of top and bottom sides on the same nanostructure linked to a surface remain unaltered. We furthermore demonstrate that the functionality of the structures remains available after an oligolysine-PEG coating is applied, enabling bioassays where functionality and stability are imperative.

Factors and methods to modulate DNA hybridization kinetics.

DNA oligonucleotides are widely used in a diverse range of research fields from analytical chemistry, molecular biology, nanotechnology to drug delivery. In these applications, DNA hybridization is often the most important enabling reaction. Achieving control over hybridization kinetics and a high yield of hybridized products is needed to ensure high-quality and reproducible results. Since DNA strands are highly negatively charged and can also fold upon itself to form various intramolecular structures, DNA hybridization needs to overcome these barriers. Nucleation and diffusion are two main kinetic limiting steps although their relative importance differs in different conditions. The effects of length and sequence, temperature, pH, salt concentration, cationic polymers, organic solvents, freezing and crowding agents are summarized in the context of overcoming these barriers. This article will help researchers in the biotechnology-related fields to better understand and control DNA hybridization, as well as provide a landscape for future work in simulation and experiment to optimize DNA hybridization systems.

A deep learning model for predicting next-generation sequencing depth from DNA sequence

Targeted high-throughput DNA sequencing is a primary approach for genomics and molecular diagnostics, and more recently as a readout for DNA information storage. Oligonucleotide probes used to enrich gene loci of interest have different hybridization kinetics, resulting in non-uniform coverage that increases sequencing costs and decreases sequencing sensitivities. Here, we present a deep learning model (DLM) for predicting Next-Generation Sequencing (NGS) depth from DNA probe sequences. Our DLM includes a bidirectional recurrent neural network that takes as input both DNA nucleotide identities as well as the calculated probability of the nucleotide being unpaired. We apply our DLM to three different NGS panels: a 39,145-plex panel for human single nucleotide polymorphisms (SNP), a 2000-plex panel for human long non-coding RNA (lncRNA), and a 7373-plex panel targeting non-human sequences for DNA information storage. In cross-validation, our DLM predicts sequencing depth to within a factor of 3 with 93% accuracy for the SNP panel, and 99% accuracy for the non-human panel. In independent testing, the DLM predicts the lncRNA panel with 89% accuracy when trained on the SNP panel. The same model is also effective at predicting the measured single-plex kinetic rate constants of DNA hybridization and strand displacement.

DNA Framework‐Engineered Long‐Range Electrostatic Interactions for DNA Hybridization Reactions

AbstractLong‐range electrostatic interactions beyond biomolecular interaction interfaces have not been extensively studied due to the limitation in engineering electric double layers in physiological fluids. Here we find that long‐range electrostatic interactions play an essential role in kinetic modulation of DNA hybridizations. Protein and gold nanoparticles with different charges are encapsulated in tetrahedral frameworks to exert diverse electrostatic effects on site‐specifically tethered single DNA strands. Using this strategy, we have successfully modulated the hybridization kinetics in both bulk solution and single molecule level. Experimental and theoretical studies reveal that long‐range Coulomb interactions are the key factor for hybridization rates. This work validates the important role of long‐range electrostatic forces in nucleic acid‐biomacromolecule complexes, which may encourage new strategies of gene regulation, antisense therapy, and nucleic acid detection.

Nanoscale Patterning of Graphene Field-Effect Transistors for Single-Molecule Functionalization and Time Series

With the discovery of low-dimensional materials, the extreme miniaturization of field-effect transistors (FETs) has revealed such devices as powerful sensors for experiments at the single-molecule scale. In particular, FET devices functionalized with an individual molecule have been able to record, via fluctuations in the electrical conductance, various types of single-molecule dynamic changes, for example enzymatic activity1 DNA hybridization2,3 or DNA folding4. So far, such studies have mostly been carried out with FETs based on 1D materials such as carbon nanotubes (CNTs) or silicon nanowires (SiNWs). The 1D geometry of these materials facilitates the capture of a 0D molecule and provides enhanced sensitivity of the conductance to the functionalized site. However, FETs based on 1D materials often present scalability issues, due to challenges in controlling their growth and/or assembly.Here, we propose an approach based on nanoscale patterning of a 2D material to assemble FET arrays compatible for single-molecule capture and detection. First, we report wafer-scale microfabrication of arrays of graphene field-effect transistors (GFETs) with micron-scale channels. Like its 1D counterpart, graphene presents a high and sensitive electrical conductivity as well as carbon-based surface chemistry for single-molecule functionalization. GFET arrays were built from high-quality graphene synthesized by chemical vapor deposition (CVD) and transferred using a custom automated process on patterned electrodes. Then, we describe the nanoscale patterning of graphene channels in nanoconstrictions suitable for single-molecule functionalization and sensitivity. Using electron-beam lithography (EBL) and deep reactive ion etching (DRIE), we were able to produce a combination of high-resolution features in graphene (<100nm) as well as nanofluidics reactions chambers (<50nm), to promote single-molecule chemistry on the graphene. We will present our recent results in functionalizing these nanoconstrictions with single-stranded DNA and in collecting high-resolution electrical time series from these constructs. Finally, we will discuss the performance of such graphene-based devices for single-molecule experiments. Bibliography Choi, Y., Weiss, G. A. & Collins, P. G. Single molecule recordings of lysozyme activity. Physical Chemistry Chemical Physics (2013). doi:10.1039/c3cp51356d Sorgenfrei, S. & Shepard, K. L. Label-free field-effect-based single-molecule detection of DNA hybridization kinetics. Nat. Nanotechnol. 6, 126–132 (2011). Vernick, S. et al. Electrostatic melting in a single-molecule field-effect transistor with applications in genomic identification. Nat. Commun. 8, 1–9 (2017). Bouilly, D. et al. Single-molecule reaction chemistry in patterned nanowells. Nano Lett. 16, 4679–4685 (2016).

DNA hybridisation kinetics using single-molecule fluorescence imaging.

Deoxyribonucleic acid (DNA) hybridisation plays a key role in many biological processes and nucleic acid biotechnologies, yet surprisingly there are many aspects about the process which are still unknown. Prior to the invention of single-molecule microscopy, DNA hybridisation experiments were conducted at the ensemble level, and thus it was impossible to directly observe individual hybridisation events and understand fully the kinetics of DNA hybridisation. In this mini-review, recent single-molecule fluorescence-based studies of DNA hybridisation are discussed, particularly for short nucleic acids, to gain more insight into the kinetics of DNA hybridisation. As well as looking at single-molecule studies of intrinsic and extrinsic factors affecting DNA hybridisation kinetics, the influence of the methods used to detect hybridisation of single DNAs is considered. Understanding the kinetics of DNA hybridisation not only gives insight into an important biological process but also allows for further advancements in the growing field of nucleic acid biotechnology.

How Surfaces Affect Hybridization Kinetics.

Hybridization between nucleic acid strands immobilized on a solid support with partners in solution is widely practiced in bioanalytical technologies and materials science. An important fundamental aspect of understanding these reactions is the role played by immobilization in the dynamics of duplex formation and disassembly. This report reviews and analyzes literature kinetic data to identify commonly observed trends and to correlate them with probable molecular mechanisms. The analysis reveals that while under certain conditions impacts from immobilization are minimal so that surface and solution hybridization kinetics are comparable, it is more typical to observe pronounced offsets between the two scenarios. In the forward (hybridization) direction, rates at the surface commonly decrease by one to two decades relative to solution, while in the reverse direction rates of strand separation at the surface can exceed those in solution by tens of decades. By recasting the deviations in terms of activation barriers, a consensus of how immobilization impacts nucleation, zipping, and strand separation can be conceived within the classical mechanism in which duplex formation is rate limited by preassembly of a nucleus a few base pairs in length, while dehybridization requires the cumulative breakup of base pairs along the length of a duplex. Evidence is considered for how excess interactions encountered on solid supports impact these processes.

Optical fiber SPR biosensor complying with a 3D composite hyperbolic metamaterial and a graphene film

In the present study, an optical fiber surface plasmon resonance (SPR) biosensor was developed for measuring time- and concentration-dependent DNA hybridization kinetics. Its design complies with a 3D Au / Al 2 O 3 multilayer composite hyperbolic metamaterial (HMM), a graphene film, and a D-shaped plastic optical fiber. According to the numerical simulation and the experimental demonstration, the SPR peak of the designed biosensor can be effectively altered in the range of visible to near-infrared by varying the HMM structure. The sensitivity of the appliance was shown to achieve a value of up to 4461 nm/RIU, allowing its applicability for bulk refractive index sensing. Furthermore, a biosensor designed in this work displayed high-resolution capability (ranging from 10 pM to 100 nM), good linearity, and high repeatability along with a detection limit down to 10 pM, thus suggesting a vast potential for medical diagnostics and clinical applications.

Evanescent wave cavity ring-down spectroscopy based interfacial sensing of prostate-specific antigen

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is implemented as a sensitive probe for the detection of prostate-specific antigen (PSA) with a sub-femtomolar limit of detection (LOD). PSA, a glycoprotein, is an essential biomarker for scrutinizing prostate cancer. Gold nanoparticles (Au NPs) were synthesized to serve as a sensitive reporter which are strongly bound to the thiol-modified target DNA. While Au NPs/target DNA flowed via a microfluidic channel, the target DNA may hybridize with aptamer DNA immobilized on the silica surface. Thus, the adsorption kinetics of hybridization between the Au NPs/target and aptamer was measured with interfacial optical absorbance using Langmuir fit of adsorption equilibrium. The LOD of Au NPs/PSA sensor reaches 0.54 fM signifying tremendous detecting proficiency of EW-CRDS. The evanescent wave is highly sensitive for small changes in absorption of material leading to a significant variation of the ring-down time constant. Moreover, Au NPs possess inherent excellent surface plasmonic properties that made them a promising probe material for the interfacial sensing process. To assess the significance of probe material, 5-carboxytetramethylrhodamine was substituted as a reporter to determine sensing kinetics, showing LOD of 99.1 fM. EW-CRDS approach could be used as an efficient sensing technique for the examination of malignant diseases and many biological reactions.

Self-Assembly of DNA-Functionalized Nanoparticles Guided by Binding Kinetics.

We use a coarse-grained model of DNA-functionalized particles (DFPs) to understand the role of DNA strand length on their self-assembly. We find that the increasing strand length for a given particle size decreases the propensity to form ordered crystalline assemblies within the simulation time. Instead, disordered structures form when the strand length exceeds a certain threshold, consistent with the previous experiments. Analysis of the simulation data based on a pair of DFPs suggests weakening interparticle interactions with increasing strand length, thereby shifting the suitable assembly conditions to lower temperatures. We find that DNA (un)hybridization kinetics at these lower temperatures becomes significantly slower, preventing systems with longer DNA strands from crystallizing successfully. We suggest that a suitable strategy to overcome this kinetic barrier is to enhance interparticle interactions for DFPs with longer DNA strands, which is achieved by increasing the DNA grafting density. We directly test this hypothesis and show successful crystallization within the simulation time for DFPs with longer strands with higher grafting densities. Our results highlight the power of computational modeling in elucidating the fundamental design principles and guiding the assembly of nanoparticles to form complex nanostructures in cases where experiments alone have not been able to do so.

Understanding DNA interactions in crowded environments with a coarse-grained model.

Nucleic acid interactions under crowded environments are of great importance for biological processes and nanotechnology. However, the kinetics and thermodynamics of nucleic acid interactions in a crowded environment remain poorly understood. We use a coarse-grained model of DNA to study the kinetics and thermodynamics of DNA duplex and hairpin formation in crowded environments. We find that crowders can increase the melting temperature of both an 8-mer DNA duplex and a hairpin with a stem of 6-nt depending on the excluded volume fraction of crowders in solution and the crowder size. The crowding induced stability originates from the entropic effect caused by the crowding particles in the system. Additionally, we study the hybridization kinetics of DNA duplex formation and the formation of hairpin stems, finding that the reaction rate kon is increased by the crowding effect, while koff is changed only moderately. The increase in kon mostly comes from increasing the probability of reaching a transition state with one base pair formed. A DNA strand displacement reaction in a crowded environment is also studied with the model and we find that rate of toehold association is increased, with possible applications to speeding up strand displacement cascades in nucleic acid nanotechnology.

Toward a Quantitative Relationship between Nanoscale Spatial Organization and Hybridization Kinetics of Surface Immobilized Hairpin DNA Probes.

Hybridization of DNA probes immobilized on a solid support is a key process for DNA biosensors and microarrays. Although the surface environment is known to influence the kinetics of DNA hybridization, so far it has not been possible to quantitatively predict how hybridization kinetics is influenced by the complex interactions of the surface environment. Using spatial statistical analysis of probes and hybridized target molecules on a few electrochemical DNA (E-DNA) sensors, functioning through hybridization-induced conformational change of redox-tagged hairpin probes, we developed a phenomenological model that describes how the hybridization rates for single probe molecules are determined by the local environment. The predicted single-molecule rate constants, upon incorporation into numerical simulation, reproduced the overall kinetics of E-DNA sensor surfaces at different probe densities and different degrees of probe clustering. Our study showed that the nanoscale spatial organization is a major factor behind the counterintuitive trends in hybridization kinetics. It also highlights the importance of models that can account for heterogeneity in surface hybridization. The molecular level understanding of hybridization at surfaces and accurate prediction of hybridization kinetics may lead to new opportunities in development of more sensitive and reproducible DNA biosensors and microarrays.

Fast and multiplexed superresolution imaging with DNA-PAINT-ERS

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

Non-Langmuir Kinetics of DNA Surface Hybridization

Hybridization of complementary single strands of DNA represents a very effective natural molecular recognition process widely exploited for diagnostic, biotechnology, and nanotechnology applications. A common approach relies on the immobilization on a surface of single-stranded DNA probes that bind complementary targets in solution. However, despite the deep knowledge on DNA interactions in bulk solution, the modeling of the same interactions on a surface are still challenging and perceived as strongly system dependent. Here, we show that a two-dimensional analysis of the kinetics of hybridization, performed at different target concentrations and probe surface densities by a label-free optical biosensor, reveals peculiar features inconsistent with an ideal Langmuir-like behavior. We propose a simple non-Langmuir kinetic model accounting for an enhanced electrostatic repulsion originating from the surface immobilization of nucleic acids and for steric hindrance close to full hybridization of the surface probes. The analysis of the kinetic data by the model enables quantifying the repulsive potential at the surface, as well as retrieving the kinetic parameters of isolated probes. We show that the strength and the kinetics of hybridization at large probe density can be improved by a three-dimensional immobilization strategy of probe strands with a double-stranded linker.

Ultrafast DNA Sensors with DNA Framework-Bridged Hybridization Reactions.

Intracellular DNA-based hybridization reactions generally occur under tension rather than in free states, which are spatiotemporally controlled in physiological conditions. However, how nanomechanical forces affect DNA hybridization efficiencies in in-vitro DNA assays, for example, biosensors or biochips, remains largely elusive. Here, we design DNA framework-based nanomechanical handles that can control the stretching states of DNA molecules. Using a pair of tetrahedral DNA framework (TDF) nanostructured handles, we develop bridge DNA sensors that can capture target DNA with ultrafast speed and high efficiency. We find that the rigid TDF handles bind two ends of a single-stranded DNA (ssDNA) and hold it in a stretched state, with an apparent stretching length comparable to its counterpart of double-stranded DNA (dsDNA) via atomic force microscopy measurement. The DNA stretching effect of ssDNA is then monitored using single-molecule fluorescence energy transfer (FRET), resulting in decreased FRET efficiency in the stretched ssDNA. By controlling the stretching state of ssDNA, we obtained significantly improved hybridization kinetics (within 1 min) and hybridization efficiency (∼98%) under the target concentration of 500 nM. The bridge DNA sensors demonstrated high sensitivity (1 fM), high specificity (single mismatch mutation discrimination), and high selectivity (suitable for the detection in serum and blood) under the target concentration of 10 nM. Controlling the stretching state of ssDNA shows great potential in biosensors, bioimaging, and biochips applications.

Probing the Mechanism of Structure-Switching Aptamer Assembly by Super-Resolution Localization of Individual DNA Molecules.

Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.

Hybridization kinetics of DNA fragments in the presence of ligands intercalating into DNA-duplexes

In this work the hybridization kinetics of DNA fragments on underlayer has been studied, when besides single-stranded DNA molecules there are also ligands in the medium that are able to intercalate into DNA-duplexes. A system of differential equations was obtained that describes the correlative change of the number of DNA-duplexes on the underlayer and the change of the number of ligands intercalating into DNA-duplexes. It was shown that the rate of underlayer filling by DNA-duplexes increases along with the enhancement of both equilibrium constant of formation reaction of DNA-duplexes and the concentration of DNA targets in the solution. It was also shown that the intercalation kinetics of ligands into DNA-duplexes relevantly depends on relation of dissociation rate constant of DNA-duplex to dissociation rate of the ligand complex with DNA-duplex. Communicated by Ramaswamy H. Sarma