Hybridization Kinetics Research Articles

Bioapplications of DNA nanotechnology at the solid-liquid interface.

DNA nanotechnology engineered at the solid-liquid interface has advanced our fundamental understanding of DNA hybridization kinetics and facilitated the design of improved biosensing, bioimaging and therapeutic platforms. Three research branches of DNA nanotechnology exist: (i) structural DNA nanotechnology for the construction of various nanoscale patterns; (ii) dynamic DNA nanotechnology for the operation of nanodevices; and (iii) functional DNA nanotechnology for the exploration of new DNA functions. Although the initial stages of DNA nanotechnology research began in aqueous solution, current research efforts have shifted to solid-liquid interfaces. Based on shape and component features, these interfaces can be classified as flat interfaces, nanoparticle interfaces, and soft interfaces of DNA origami and cell membranes. This review briefly discusses the development of DNA nanotechnology. We then highlight the important roles of structural DNA nanotechnology in tailoring the properties of flat interfaces and modifications of nanoparticle interfaces, and extensively review their successful bioapplications. In addition, engineering advances in DNA nanodevices at interfaces for improved biosensing both in vitro and in vivo are presented. The use of DNA nanotechnology as a tool to engineer cell membranes to reveal protein levels and cell behavior is also discussed. Finally, we present challenges and an outlook for this emerging field.

Single-Molecule Kinetic Fingerprinting for the Ultrasensitive Detection of Small Molecules with Aptasensors.

Aptamers have emerged as promising molecular tools for small-molecule analyte sensing. However, the performance of such aptasensors is generally limited by leakage since it has been difficult to completely suppress signal in the absence of analyte, resulting in a compromise between sensitivity and specificity. Here, we describe a methodology for the ultrasensitive detection of analytes combining aptasensors with single-molecule kinetic fingerprinting. A short, fluorescently labeled DNA probe is utilized to detect the structural changes upon ligand binding to the designed hairpin-shaped aptasensor probe. The Poisson statistics of binding and dissociation events of the DNA probe to single surface-immobilized aptasensor molecules is monitored by total internal reflection fluorescence microscopy, permitting the high-accuracy discrimination of the ligand bound and ligand-free states, resulting in zero background. The programmable dynamics of the hairpin enables fine-tuning of the hybridization kinetics of the fluorescent probe, rendering the acquisition time sufficiently flexible to optimize discrimination. Remarkable detection limits are achieved for a diverse set of analytes when spiked into chicken meat extract: the nucleotide adenosine (0.3 pM), the insecticide acetamiprid (0.35 pM), and the dioxin-like toxin PCB-77 (0.72 pM), which is superior to recently reported aptasensors. Our generalizable method significantly improves the performance of aptasensors, with the potential to extend to other molecular biomarkers.

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy.

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.

Kinetics of Strand Displacement and Hybridization on Wireframe DNA Nanostructures: Dissecting the Roles of Size, Morphology, and Rigidity.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

Increasing target hybridization kinetics by immobilizing DNA probes at Au nanoflower ultramicroelectrode

Although different electrochemical DNA sensors have been widely developed for analytical purpose, the effective way to immobilize DNA probes on basic electrode surface for increasing their binding rate with target is rarely reported. Herein, a new scheme for increasing DNA probes-target hybridization kinetics using gold nanoflower ultramicroelectrode (UME) as DNA probe immobilization surface was developed. In details, the ATP aptamer was selected as the model to demonstrate this E-DNA sensor’s properties. Our results showed that the DNA aptamer/ATP hybridization rate obtained at gold nanoflower UME was about 10-fold and 4-fold greater than that obtained at the macroelectrode surface, and even in the homogeneous solution, respectively. This increase of DNA aptamer hybridization rate was confirmed to be related with the smaller size of UME and the specific structure of gold nanoflower.

Multiplexed assessment of the surface density of DNA probes on DNA microarrays by surface plasmon resonance imaging

In terms of hybridization assays surface plasmon resonance imaging (SPRi) offers high throughput, label-free and real-time monitoring of the binding kinetics. This requires DNA microarrays on bare or modified gold SPRi chips, which are generally premade by an off-line microspotting procedure. Therefore, the surface density of the immobilized probes is not known although it is an essential quality control parameter, especially, when it can vary in a broad range as in case of self-assembled thiol-labeled DNAs on gold surface. Here we show that the small molecular weight ruthenium(III) hexamine complex (RuHex) introduced earlier for electrochemical quantitation of DNA coverage on gold electrodes can be used also in SPRi to assess the surface density of DNA probes in DNA microarrays. A single injection of RuHex solution allows the simultaneous visualization and quantification of the surface density of DNA probes (ranging in this study from 4 × 1011 to 1.7 × 1013 molecules cm−2) on all spots of a microarray made by microspotting thiol labeled short DNA probes both in prehybridized and single-stranded form on a gold SPRi chip. The methodology was applied to determine the effect of the surface density of DNA probes on the hybridization efficiency and kinetics of complementary microRNAs, using hsa-miR-208a-3p as model. Single mismatch duplexes were found to be more effectively destabilized than fully complementary duplexes by steric hindrance at large surface densities of the DNA probes, which offers an effective mean to increase single mismatch selectivity.

Overhang molecular beacons encapsulated in tethered cationic lipoplex nanoparticles for detection of single-point mutation in extracellular vesicle-associated RNAs

Detection of specific extracellular RNAs has been developed for non-invasive cancer diagnosis. However, accurate and efficient identification of RNAs with single-point mutation in cancer cells-derived extracellular vesicles (EVs) is challenging. Herein, we present a unique overhang molecular beacon with internal dye (Ohi-MB) with a stable hairpin structure, fast hybridization kinetics and single mismatch specificity. Ohi-MBs are encapsulated in cationic lipoplex nanoparticles (CLNs) that are tethered on a gold coated glass slide as a chip, which can capture circulating EVs and detect encapsulated target RNAs in-situ in a single step. The capability of detection of single-point mutation by CLN-Ohi-MB is demonstrated in artificial EVs and cancer cells. This CLN-Ohi-MB biochip could quantify single-point mutations in KRAS mRNA (G12C, G12D, G12V) in pancreatic cancer cell-derived EVs and single-point mutations in EGFR mRNA (L858R and T790M) in lung cancer cell-derived EVs with high specificity, not achievable by conventional molecular probes. We show that CLN-Ohi-MB biochip could selectively and sensitively identify single-point mutations in KRAS mRNA in human serum EVs, distinguishing pancreatic cancer patients with different mutations.

Framework Nucleic Acid-Mediated Pull-Down MicroRNA Detection with Hybridization Chain Reaction Amplification.

Gastric cancer remains a disease of high mortality worldwide due to its poor prognosis. Previous studies have shown that microRNAs (miRNAs) are effective biomarkers for early diagnosis of gastric cancer. To realize sensitive detection of related miRNAs for improved early diagnosis, classification, and survival prognosis of gastric cancer, herein we developed a framework nucleic acid (FNA)-mediated microarray for quantitative analysis of multiple miRNAs. By rationally designing FNA with different sizes, we systematically modulated the surface density and lateral interactions of DNA probes, which provides an effective means for programmable tailoring of the hybridization efficiency and kinetics of the biosensing interface. We found that the hybridization efficiency was increased along with the size of the FNA and was optimum for FNA-17. In combination with the hybridization chain reaction amplification strategy, this established FNA microarray can serve as an ultrasensitive and selective analytical platform for simultaneous multiplexed detection of miRNA (e.g., FNA-miR-652, FNA-miR-627, and FNA-miR-629) biomarkers in gastric cancer.

Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices

We describe a tethered multifluorophore motion assay based on DNA origami for revealing bimolecular reaction kinetics on the single-molecule level. Molecular binding partners may be placed at user-defined positions and in user-defined stoichiometry; and binding states are read out by tracking the motion of quickly diffusing fluorescent reporter units. Multiple dyes per reporter unit enable singe-particle observation for more than 1 hour. We applied the system to study in equilibrium reversible hybridization and dissociation of complementary DNA single strands as a function of tether length, cation concentration, and sequence. We observed up to hundreds of hybridization and dissociation events per single reactant pair and could produce cumulative statistics with tens of thousands of binding and unbinding events. Because the binding partners per particle do not exchange, we could also detect subtle heterogeneity from molecule to molecule, which enabled separating data reflecting the actual target strand pair binding kinetics from falsifying influences stemming from chemically truncated oligonucleotides. Our data reflected that mainly DNA strand hybridization, but not strand dissociation, is affected by cation concentration, in agreement with previous results from different assays. We studied 8-bp-long DNA duplexes with virtually identical thermodynamic stability, but different sequences, and observed strongly differing hybridization kinetics. Complementary full-atom molecular-dynamics simulations indicated two opposing sequence-dependent phenomena: helical templating in purine-rich single strands and secondary structures. These two effects can increase or decrease, respectively, the fraction of strand collisions leading to successful nucleation events for duplex formation.

Identification of Individual Immobilized DNA Molecules by Their Hybridization Kinetics Using Single-Molecule Fluorescence Imaging.

Single-molecule fluorescence methods can count molecules without calibration, measure kinetics at equilibrium, and observe rare events that cannot be detected in an ensemble measurement. We employ total internal reflection fluorescence microscopy to monitor hybridization kinetics between individual spatially resolved target DNA molecules immobilized at a glass interface and fluorescently labeled complementary probe DNA in free solution. Using super-resolution imaging, immobilized target DNA molecules are located with 36 nm precision, and their individual duplex formation and dissociation kinetics with labeled DNA probe strands are measured at site densities much greater than the diffraction limit. The purpose of this study is to evaluate uncertainties in identifying these individual target molecules based on their duplex dissociation kinetics, which can be used to distinguish target molecule sequences randomly immobilized in mixed-target samples. Hybridization kinetics of individual target molecules are determined from maximum likelihood estimation of their dissociation times determined from a sample of hybridization events at each target molecule. The dissociation time distributions thus estimated are sufficiently narrow to allow kinetic discrimination of different target sequences. For example, a single-base thymine-to-guanine substitution on immobilized strands produces a 2.5-fold difference in dissociation rates of complementary probes, allowing for the identification of individual target DNA molecules by their dissociation rates with 95% accuracy. This methodology represents a step toward high-density single-molecule DNA microarray sensors and a powerful tool to investigate the kinetics of hybridization at surfaces at the molecular level, providing information that cannot be acquired in ensemble measurements.

Probing Multiple Binding Modes of DNA Hybridization: A Comparison between Single-Molecule Observations and Ensemble Measurements.

Interactions between biomolecules are generally analyzed by ensemble measurements, assuming that the interactions occur in a single binding manner. However, such interactions may occur via multiple binding modes. We investigated the kinetics of DNA hybridization as a multiple dynamic model of biomolecular interactions. Two kinetic analyses were performed with a single-molecule observation using total internal reflection fluorescence microscopy (TIRFM) and with ensemble measurements using a quartz-crystal microbalance (QCM) biosensor. We observed the DNA hybridization of 8 and 12 bp DNAs with random sequences and dA12–dT12 and calculated the kinetic parameters, including the dissociation rate constant (koff). Hybridization of 8 bp DNA proceeded mainly via a single binding mode. However, hybridization of 12 bp DNA indicated at least two different binding modes and dA12–dT12 hybridization showed multiple binding modes. For the multiple binding interactions, the kinetic parameters obtained from TIRFM and QCM were different because kinetic parameters obtained from QCM indicate average number of molecules, whereas those from TIRFM indicate average association time. The present study revealed the details of multiple interactions, which can be utilized for better understanding of not only DNA hybridization but also biomolecular interaction mechanisms.

Measuring Oligonucleotide Hybridization Kinetics in Solution using a time-Resolved 3D Single-Molecule Tracking Technique

Single-molecule measurements of DNA hybridization kinetics are mostly performed on a surface or inside a trap. Here we demonstrate a time-resolved, 3D single-molecule tracking (3D-SMT) method that allows us to follow a freely diffusing ssDNA molecule in solution for hundreds of milliseconds or even seconds and observe multiple annealing and melting events taking place on the same molecule. This is achieved by combining confocal-feedback 3D-SMT with time-domain fluorescence lifetime measurement, where fluorescence lifetime serves as the indicator of hybridization. With sub-diffraction-limit spatial resolution in molecular tracking and 15 ms temporal resolution in monitoring the change of reporter's lifetime, we have demonstrated a full characterization of annealing rate (kon = 5.13 × 106 M−1 s−1), melting rate (koff = 9.55 s−1), and association constant (Ka = 0.54 μM−1) of an 8 bp duplex model system diffusing at 4.8 μm2 s−1. As our method completely eliminates the photobleaching artifacts and diffusion interference, our kon and koff results well represent the real kinetics in solution. Our binding kinetics measurement can be carried out in a low signal-to-noise ratio condition (SNR ≈ 1.4) where ∼130 recorded photons are sufficient for a lifetime estimation. Using a population-level analysis, we can characterize hybridization kinetics over a wide range (0.5-125 s−1), even beyond the reciprocals of the lifetime monitoring temporal resolution and the average track duration. To apply this technique to study hybridization kinetics in live cells, we are testing various oxygen scavenger systems that are live-cell compatible. Our preliminary results have shown improved dye stability in our tracking experiments.

Influence of thermodynamically unfavorable secondary structures on DNA hybridization kinetics.

Nucleic acid secondary structure plays an important role in nucleic acid–nucleic acid recognition/hybridization processes, and is also a vital consideration in DNA nanotechnology. Although the influence of stable secondary structures on hybridization kinetics has been characterized, unstable secondary structures, which show positive ΔG° with self-folding, can also form, and their effects have not been systematically investigated. Such thermodynamically unfavorable secondary structures should not be ignored in DNA hybridization kinetics, especially under isothermal conditions. Here, we report that positive ΔG° secondary structures can change the hybridization rate by two-orders of magnitude, despite the fact that their hybridization obeyed second-order reaction kinetics. The temperature dependence of hybridization rates showed non-Arrhenius behavior; thus, their hybridization is considered to be nucleation limited. We derived a model describing how ΔG° positive secondary structures affect hybridization kinetics in stopped-flow experiments with 47 pairs of oligonucleotides. The calculated hybridization rates, which were based on the model, quantitatively agreed with the experimental rate constant.

Precision Tuning of DNA- and Poly(ethylene glycol)-Based Nanoparticles via Coassembly for Effective Antisense Gene Regulation.

Development of safe and effective delivery vectors remains a critical challenge for oligonucleotides-based biopharmaceuticals. In this work, we incorporate antisense oligonucleotides into micellar nanoparticles via co-assembly of two block copolymers, DNA-b-poly(ɛ-caprolactone) and poly(ethylene glycol)-b-poly(ɛ-caprolactone), and the hydrophobic homopolymer, poly(ɛ-caprolactone). By fine-tuning the spacing of particle surface moieties through adding a diluent homopolymer to the micellar core, we conduct a systematic investigation of structure-property correlations among hybridization kinetics, nuclease resistance, cellular uptake, and antisense efficacy. It is found that, when an intermediate density is achieved, nuclease access of the DNA is greatly hindered, but formation of double-stranded DNA is nearly unaffected. Remarkably, micelles showing the highest selectivity (good nuclease resistance while still maintaining hybridization kinetics) also exhibit the highest antisense gene silencing efficacies. ...

Predicting DNA hybridization kinetics from sequence.

Hybridization is a key molecular process in biology and biotechnology, but to date there is no predictive model for accurately determining hybridization rate constants based on sequence information. Here we report a weighted neighbor voting (WNV) prediction algorithm, in which the hybridization rate constant of an unknown sequence is predicted based on similarity reactions with known rate constants. To construct this algorithm we first performed 210 fluorescence kinetics experiments to observe the hybridization kinetics of 100 different DNA target and probe pairs (36nt subsequences of the CYCS and VEGF genes) at temperatures ranging from 28°C to 55°C. Automated feature selection and weighting optimization resulted in a final 6-feature WNV model, which can predict hybridization rate constants of new sequences to within a factor of 3 with ≈91% accuracy, based on leave-one-out cross-validation. Accurate prediction of hybridization kinetics allows design of efficient probe sequences for genomics research.

Design and Characterization of a Singly Labeled Fluorescent Smart Probe for In Vitro Detection of miR-21.

A sensitive hairpin smart probe (SP) has been developed and tested for its sequence-specificity and sensitivity for detecting microRNAs (miRNAs). The loop sequence of this SP is perfectly complementary to microRNA-21 (miR-21) sequence. This miRNA regulates certain biological processes and has been implicated in certain forms of cancer. The stem of the new SP consists of a fluorophore on one end and multiple guanine bases on the opposing end are used as quenchers. The fluorescence of the SP is significantly quenched by the guanine bases at room temperature and in the absence of the miR-21 target. The presence of miR-21 switches on the fluorescence due to spontaneous hybridization of the SP with this target, which also forces the stem hybrid of the SP apart. This new SP successfully discriminated between the perfect miR-21 target and two closely similar single-base mismatch sequences. When the SP was incubated with the miR-21 at 37 ℃, the hybridization kinetics increased seven times, compared to room temperature hybridization. Overall, this new SP shows good detection sensitivity and gives a limit of detection and limit of quantitation of 14.0 nM and 46.7 nM, respectively. This detection platform represents a simple, fast, mix-and-read homogeneous assay for sequence-specific detection of miR-21, and it can be adapted for other related diagnostic applications.

2'-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA.

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2′-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.

2'-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA.

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2′-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.

Biomolecular Steric Hindrance Effects Are Enhanced on Nanostructured Microelectrodes.

The availability of rapid approaches for quantitative detection of biomarkers would drastically impact global health by enabling decentralized disease diagnosis anywhere that patient care is administered. A promising new approach, the electrochemical steric hindrance hybridization assay (eSHHA) has been introduced for quantitative detection of large proteins (e.g., antibodies) with a low nanomolar detection limit within 10 min. Here, we report the use of a nanostructured microelectrode (NME) platform for eSHHA that improves the performance of this approach by increasing the efficiency and kinetics of DNA hybridization. We demonstrated that eSHHA on nanostructured microelectrodes leverages three effects: (1) steric hindrance effects at the nanoscale, (2) a size-dependent hybridization rate of DNA complexes, and (3) electrode morphology-dependent blocking effects. As a proof of concept, we showed that the sensitivity of eSHHA toward a model antibody is enhanced using NMEs as scaffolds for this reaction. We improved the detection limit of eSHHA, taking advantage of nanostructured surfaces to allow the use of longer capture strands for detection of proteins. Finally, we concluded that using the eSHHA approach in conjunction with nanostructured microelectrodes is an advantageous alternative to conventional macroelectrodes as the sensitivity and detection limits are enhanced.

DNA-Driven Assembly: From Polyhedral Nanoparticles to Proteins

Directed crystallization of a large variety of nanoparticles, including proteins, via DNA hybridization kinetics has led to unique materials with a broad range of crystal symmetries. The nanoparticles are functionalized with DNA chains that link neighboring functionalized units. The shape of the nanoparticle, the DNA length, the sequence of the hybridizing DNA linker, and the grafting density determine the crystal symmetries and lattice spacing. By carefully selecting these parameters, one can, in principle, achieve all the symmetries found for both atomic and colloidal crystals of asymmetric shapes as well as new symmetries and can drive transitions between them. A scale-accurate coarse-grained model with explicit DNA chains provides the design parameters, including the degree of hybridization, to achieve specific crystal structures. The model also provides surface energy values to determine the shape of defect-free single crystals with macroscopic anisotropic properties, which has potential for the fabrication of materials with specific optical and mechanical properties.