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Accelerated DNA hybridization chain reactions (HCRs) using DNA origami as a scaffold have received considerable attention in dynamic DNA nanotechnology. However, tailor-made designs are essential for DNA origami scaffolds, hampering the practical application of accelerated HCRs. Here, we constructed the semilocalized HCR and localized HCR systems using magnetic beads (MBs) as a simple scaffold to explore them for the enzyme-free miR-21 detection. The semilocalized HCR system relied on free diffusing one hairpin DNA and MBs immobilized with another hairpin DNA, and the localized HCR system relied on MBs coimmobilized with two hairpin DNAs. We demonstrated that the DNA density on MBs plays a critical role in HCR kinetics and limit of detection (LOD). Among semilocalized HCR systems, MBs with a medium DNA density showed a faster HCR and lower LOD (10 pM) than the diffusive (conventional) HCR system (LOD: 86 pM). In contrast, the HCR further accelerated for the localized HCR systems as the DNA density increased. The localized HCR system with the highest DNA density showed the fastest HCR and the lowest LOD (533 fM). These findings are of great importance for the rational design of accelerated HCRs using simple scaffolds for practical applications.

Long-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.

Characterization of DNA at solid/liquid interfaces remains a challenge because most surface-sensitive techniques are unable to provide quantitative insight into the base content, length, or structure. Surface-enhanced Raman scattering measurements of DNA hybridization on plasmonic-metal substrates have been used to overcome small Raman-scattering cross-sections; however, surface-enhanced Raman spectroscopy measurements are not generally quantitative due to the fall-off in the scattering signal with the decay of the electric field enhancement from the surface, which also limits the length of oligonucleotides that can be investigated. In this work, we introduce an experimental methodology in which confocal Raman microscopy is used to characterize hybridization reactions of ssDNA immobilized at the solid/liquid interface of porous silica particles. By focusing the femtoliter confocal probe volume within a single porous particle, signal enhancement arises from the ∼1500-times greater surface area detected compared to a planar substrate. Because the porous support is a purely dielectric material, the scattering signal is independent of the proximity of the oligonucleotide to the silica surface. With this technique, we characterize a 19-mer capture strand and determine its hybridization efficiency with 9-mer and 16-mer target sequences from the scattering of a structurally insensitive phosphate-stretching mode. Changes in polarizability and frequency of scattering from DNA bases were observed, which are consistent with Watson-Crick base pairing. Quantification of base content from their duplex scattering intensities allows us to discriminate between hybridization of two target strands of equivalent length but with different recognition sequences. A duplex having a single-nucleotide polymorphism could be distinguished from hybridization of a fully complementary strand based on differences in base content and duplex conformation.

Studying the DNA hybridization equilibrium via brute force molecular dynamics (MD) or commonly used advanced sampling approches is notoriously difficult at atomistic lengthscale. However, besides providing a more realistic modeling of this microscopic phenomenon, atomistic resolution is a necessity for some fundamental research questions, such as the ones related to DNA’s chirality. Here, we describe an order parameter-based advanced sampling technique to calculate the free energy surface of hybridization and estimate melting temperature of DNA oligomers at atomistic resolution, using a native topology-based order parameter. We show that the melting temperatures estimated from our atomistic simulations follow an order consistent with the predictions from melting experiments and those from the nearest neighbor model, for a range of DNA sequences of different GC content. Moreover, free energy surfaces and melting temperatures are calculated to be identical for D- and L-enantiomers of Drew-Dickerson dodecamer.Abstract FigureGraphical TOC Entry

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5–15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture 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.

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.

Single-molecule Forster Resonance Energy Transfer was used to observe the adsorption of fluorescently-labeled “target” DNA oligonucleotides and their association and hybridization with complementary DNA “probes” tethered to the surface as a function of surface grafting density. Ionic strength was varied systematically to disentangle the potentially competing effects of probe accessibility and electrostatic repulsion. At high ionic strength, when the Debye length was ~1 nm, the adsorption of target DNA was not significantly inhibited by the presence of tethered probe DNA, even at high grafting density, and the fraction of adsorbed target strands undergoing hybridization increased systematically with grafting density, leading to a dramatic increase in the net hybridization rate at high grafting density. However, at lower ionic strength, when the Debye length was ≥3 nm, the adsorption rate of target DNA decreased and the fraction of adsorbed target strands undergoing hybridization saturated at high probe grafting density (≥7,000 strands/µm2), presumably due to electrostatic repulsion. As a result, the net rate of hybridization exhibited a maximum as a function of grafting density. This has important consequences for the design of systems that optimize surface-mediated DNA hybridization under low-salt high-stringency conditions.

Hybridization chain reaction (HCR) was a significant discovery for the development of nanoscale materials and devices. One key challenge for HCR is the vulnerability to background leakage in the absence of the initiator. Here, we systematically analyze the sources of leakage and refine leak-resistant rule by using molecular thermodynamics and dynamics, biochemical and biophysical methods. Transient melting of DNA hairpin is revealed to be the underlying cause of leakage and that this can be mitigated through careful consideration of the sequence thermodynamics. The transition threshold of the energy barrier is proposed as a testing benchmark of leak-resistance DNA hairpins. The universal design of DNA hairpins is illustrated by the analysis of hsa-miR-21-5p as biomarker when used in conjunction with surface-enhanced Raman spectroscopy. We further extend the strategy for specific signal amplification of miRNA homologs. Significantly, it possibly provides a practical route to improve the accuracy of DNA self-assembly for signal amplification, and that could facilitate the development of sensors for the sensitive detection of interest molecules in biotechnology and clinical medicine.

By assembling DNAzyme on DNA nanowires through DNA hybridization, we have developed a novel accelerated DNAzyme-based fluorescent nanoprobe for fast, sensitive and selective detection of miRNA. Moreover, the strategy was successfully applied for in situ imaging of miRNA-21 in different cell lines.

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2+ cancer cells using the anti‐HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2+ cells in a background of HER2− cells and peripheral blood leukocytes, which remained almost non‐fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC., Cancer cell detection via an amplified fluorescence signal: We developed a programmable DNA circuit system based on hybridization chain reaction (HCR); this is a bioinspired signal amplification approach that allows specific and sensitive detection of HER2+ breast cancer cells among HER2− cells, including freshly isolated peripheral blood mononuclear cells.

A robust understanding of the sequence-dependent thermodynamics of DNA hybridization has enabled rapid advances in DNA nanotechnology. A fundamental understanding of the sequence-dependent kinetics and mechanisms of hybridization and dehybridization remains comparatively underdeveloped. In this work, we establish new understanding of the sequence-dependent hybridization/dehybridization kinetics and mechanism within a family of self-complementary pairs of 10-mer DNA oligomers by integrating coarse-grained molecular simulation, machine learning of the slow dynamical modes, data-driven inference of long-time kinetic models, and experimental temperature-jump infrared spectroscopy. For a repetitive ATATATATAT sequence, we resolve a rugged dynamical landscape comprising multiple metastable states, numerous competing hybridization/dehybridization pathways, and a spectrum of dynamical relaxations. Introduction of a G:C pair at the terminus (GATATATATC) or center (ATATGCATAT) of the sequence reduces the ruggedness of the dynamics landscape by eliminating a number of metastable states and reducing the number of competing dynamical pathways. Only by introducing a G:C pair midway between the terminus and the center to maximally disrupt the repetitive nature of the sequence (ATGATATCAT) do we recover a canonical "all-or-nothing" two-state model of hybridization/dehybridization with no intermediate metastable states. Our results establish new understanding of the dynamical richness of sequence-dependent kinetics and mechanisms of DNA hybridization/dehybridization by furnishing quantitative and predictive kinetic models of the dynamical transition network between metastable states, present a molecular basis with which to understand experimental temperature jump data, and furnish foundational design rules by which to rationally engineer the kinetics and pathways of DNA association and dissociation for DNA nanotechnology applications.

With the continuous development of DNA nanotechnology, various spatial DNA structures and assembly techniques emerge. Hybridization chain reaction (HCR) is a typical example with exciting features and bright prospects in biosensing, which has been intensively investigated in the past decade. In this Spotlight on Applications, we summarize the assembly principles of conventional HCR and some novel forms of linear/nonlinear HCR. With advantages like great assembly kinetics, facile operation, and an enzyme-free and isothermal reaction, these strategies can be integrated with most mainstream reporters (e.g., fluorescence, electrochemistry, and colorimetry) for the ultrasensitive detection of abundant targets. Particularly, we select several representative studies to better illustrate the novel ideas and performances of HCR strategies. Theoretical and practical utilities are confirmed for a range of biosensing applications. In the end, a deep discussion is provided about the challenges and future tasks of this field.

Ten mesorhizobial strains isolated from root-nodules of Anthyllis vulneraria by trapping using soils from southern France were studied to resolve their taxonomy. Their 16S rDNA sequences were identical and indicated that they are affiliated to the genus Mesorhizobium within the group M. prunaredense/M. delmotii/M. temperatum/M. mediterraneum/M. wenxiniae and M. robiniae as the closest defined species. Their evolutionary relationships with validated species were further characterized by multilocus sequence analysis (MLSA) using 4 protein-coding housekeeping genes (recA, atpD, glnII and dnaK), that divides the strains in two groups, and suggest that they belong to two distinct species. These results were well-supported by MALDI-TOF mass spectrometry analyses, wet-lab DNA-DNA hybridization (≤58%), and genome-based species delineation methods (ANI 96%, in silico DDH 70%), confirming their affiliation to two novel species. Based on these differences, Mesorhizobium ventifaucium (STM4922

Coronavirus (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as a deadly pandemic. The genomic analysis of SARS-CoV-2 is performed using a reverse transcription-polymerase chain reaction (RT-PCR) technique for identifying viral ribonucleic acid (RNA) in infected patients. However, the RT-PCR diagnostic technique is manually laborious and expensive; therefore, it is not readily accessible in every laboratory. Methodological simplification is crucial to combat the ongoing pandemic by introducing quick, efficient, and affordable diagnostic methods. Here, we report how microcantilever sensors offer promising opportunities for rapid COVID-19 detection. Our first attempt was to capture the single-stranded complementary DNA of SARS-CoV-2 through DNA hybridization. Therefore, the microcantilever surface was immobilized with an oligonucleotide probe and detected using complementary target DNA hybridization by a shift in microcantilever resonance frequency. Our results show that microcantilever sensors can discriminate between complementary and noncomplementary target DNA on a micro to nanoscale. Additionally, the microcantilever sensors’ aptitude toward partial complementary DNA determines their potential to identify new variants of coronavirus. Therefore, microcantilever sensing could be a vital tool in the effort to extinguish the spreading COVID-19 pandemic.

DNA-directed nanoparticle (DNA–NP) systems provide various applications in sensing, medical diagnosis, data storage, plasmonics and photovoltaics. Bonding probability and melting properties are helpful to evaluate the selectivity, thermostability and thermosensitivity of these applications. We investigated the influence of temperature, nanoparticle size, DNA chain length and surface grafting density of DNA on one nanoparticle on the DNA dynamic hybridization percentage and melting properties of DNA–NP assembly systems by molecular dynamics simulation. The high degree of consistency of free energy estimations for DNA hybridization via our theoretical deduction and the nearest-neighbor rule generally used in experiments validates reasonably our DNA model. The melting temperature and thermosensitivity parameter are determined by the sigmoidal melting curves based on hybridization percentage versus temperature. The results indicated that the hybridization percentage presents a downward trend with increasing temperature and nanoparticle size. Applications based on DNA–NP systems with bigger nanoparticle size, such as DNA probes, have better selectivity, thermostability and thermosensitivity. There exist optimal DNA chain length and surface grafting density where the hybridization percentage reaches the maximal value. The melting temperature reaches a maximum at the point of optimal grafting density, while the thermosensitivity parameter presents an upward trend with the increase of grafting density. Several physical quantities consisting of the radial density function, root mean square end-to-end distance, contact distance parameter and effective volume fraction are used to analyse DNA chain conformations and DNA–NP packing in the assembly process. Our findings provide the theoretical basis for the improvement and optimization of applications based on DNA–NP systems.

Sensitive and specific DNA hybridization is essential for nucleic acid chemistry. Competitive composition of probe and blocker has been the most adopted probe design for its relatively high sensitivity and specificity. However, the sensitivity and specificity were inversely correlated over the length and concentration of the blocker strand, making the optimization process cumbersome. Herein, we construct a theoretical model for competitive DNA hybridization, which disclose that both the thermodynamics and kinetics contribute to the inverse correlation. Guided by this, we invent the 4-way Strand Exchange LEd Competitive DNA Testing (SELECT) system, which breaks up the inverse correlation. Using SELECT, we identified 16 hot-pot mutations in human genome under uniform conditions, without optimization at all. The specificities were all above 140. As a demonstration of the clinical practicability, we develop probe systems that detect mutations in human genomic DNA extracted from ovarian cancer patients with a detection limit of 0.1%., Optimisation of nucleic acid probes and blocker strands can be laborious. Here the authors construct a theoretical model of competitive DNA hybridisation to design DNA probes for optimisation-free mutation detection.

Single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids without the need for synchronization of molecular states, but the current experiments are not carried out in a native cellular context. Here we describe an integrated 3D single-molecule tracking and lifetime measurement method that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. By comparing the hybridization kinetics of the same DNA strand in vitro, we found the association constants can be 13- to 163-fold higher in the molecular crowding cellular environment.

Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in label-free DNA detection. Owing to the limitation in chain length, it is however still challenging for SERS as a routine method to explore the intrinsic structural information on unmodified DNA. Here, we develop a universal SERS-based approach toward quantification of A/G in single-stranded DNAs (12 up to 28 bases) by introducing a novel interfacial agent, dichloromethane. DNA hybridization is successfully probed as evidenced by the typical SERS bands attributed to hydrogen bonds in a hairpin structure. More importantly, enlarged space of "hot spots" in SERS enables discrimination of single-base mutation in double-stranded DNA with 100 bases, which as a proof-of-concept study will pave a new avenue for highly sensitive DNA detection in clinical applications.

An automated DNA hybridization transfer in a microflow reactor is demonstrated by moving paramagnetic beads between two spatially separate solutions with different pH values. The microbeads-based microfluidic platform is fully automated and programmable. It employs a robust chemical procedure for specific DNA hybridization transfer in microfluidic devices under isothermal conditions based on reversible pH alterations. The method takes advantage of high-speed DNA hybridization and denaturation on beads under flow conditions, high fidelity of DNA hybridization, and small sample volumes. The microfluidic platform presented is saleable and applicable to many areas of modern biotechnology such as DNA hybridization chip microarrays, molecular computation, on-chip selection of functional nucleic acids, high-throughput screening of chemical libraries for drug discovery, and DNA amplification and sequencing.

A key challenge in many biological studies is the inability to control the placement of cells in two and three dimensions. As our understanding of the importance of complexity in cellular communities increases, better tools are needed to control the spatial arrangements of cells. One universal method to govern these interactions is DNA hybridization, which relies on the inherent interaction between complementary DNA sequences. DNA hybridization has long been used to assemble complex structures of nanoparticles and more recently has been applied to the complex arrangements of cells. Using this technology, our understanding of biological interactions has significantly improved. Improvement of methods to control the interactions between cells provides powerful tools to test hypotheses about intercellular interactions, nutrient transfer, and complex diseases.

A Gram-stain-positive, rod-shaped, motile bacterial strain, designated 3-2-2T, was isolated from field topsoil collected from a western suburb of Nanyang city, Henan province, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 3-2-2T was a member of the genus Bacillus and most closely related to Bacillus fortis R-6514T (98.9 % similarity), Bacillus terrae RA9T (98.0 %) and Bacillus fordii R-7190T (97.7 %). A draft genome sequence determined for strain 3-2-2T revealed a DNA G+C content of 42.2 mol%. The average nucleotide identity and digital DNA–DNA hybridization values between 3-2-2T and the closely related Bacillus species ranged 79.4–84.2 % and 23.4–24.6 %. The major fatty acids of strain 3-2-2T were iso-C15 : 0, anteiso-C15 : 0, iso-C14 : 0 and iso-C16 : 0. The major isoprenoid quinone was MK-7. meso-Diaminopimelic acid was detected in the peptidoglycan. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and an unidentified lipid. The results of phylogenetic analyses, in silico genomic comparisons, and chemotaxonomic and phenotypic analyses clearly indicated that strain 3-2-2T represents a novel species within the genus Bacillus , for which the name Bacillus acidinfaciens sp. nov. is proposed. The type strain is 3-2-2T (=CGMCC 1.13685T=LMG 30839T).

Sensitive detection of microRNAs (miRNAs) that serve as a disease marker could advance the diagnosis and treatment of diseases. Many methods used for quantitative detection of miRNAs, such as PCR-based approaches or the hybridization chain reaction, have presented challenges due to the complicated and time-consuming-procedures that are required. In this manuscript, a simple triggerable mutually amplified signal (TMAS) probe was designed and enriched within the center of a microfluidic chip and then used for one-step quantitative detection of microRNAs via surface enhanced Raman scattering (SERS) technology. First, many mutually amplified double strands are produced via an enzyme-free target-strand displacement recycling reaction initiated by the target miRNA, that result in the generation of an enhanced SERS signal. Second, microfluidic chips that utilize alternating current (AC) electrokinetic flow technology produce efficient mixing and rapid concentration to improve the DNA hybridization rate and further enhance the SERS signal intensity. This method enables the sensitive and rapid detection of miR-21 in human breast cancer cells within 30 min with a detection limit of 2.33 fM. Compared with traditional methods, this novel method overcomes the shortcomings resulting from complex operations, and has the advantages of high sensitivity, short assay time, and reduced sample usage.

Here we report a bacteria detection method based on a flow cytometric bead system and 16S rRNA-targeted oligonucleotide probes. Polymerase chain reaction (PCR) was first used to acquire bacterial DNA including bacteria-specific sequences. Half of the resulting target DNA was then captured by a capture probe immobilized on a magnetic microbead (MB) surface. The other half of the target DNA was hybridized with a fluorescence-labeled signal probe. In this manner, a sandwich DNA hybridization involving a MB-based capture probe, the target DNA, and a signal probe was realized. The MB carriers modified with reporter dye were analyzed one by one by flow cytometry through a capillary. Using PCR amplicons and this flow cytometric bead system, a detection limit of 180 cfu mL−1 was achieved, along with high selectivity that permitted the discrimination of different targets when challenged with control bacteria targets and multiplexing capabilities that enabled the simultaneous detection of two kinds of bacteria. Given these advantages, the developed method can be used for the highly sensitive and specific PCR amplicon analysis of DNA extracted from a fresh bacterial culture, as well as multiplex target analysis.

In his study, we report a fluorescence method for homogeneous detection of influenza A (H1N1) DNA sequence based on G-quadruplex-NMM complex and assistance-DNA (A-DNA) inhibition. The quadruplex-based functional DNA (QBF-DNA), composed of a complementary probe to the target H1N1 DNA sequence and G-rich fragment, was designed as the signal DNA. The A-DNA consisted of two parts, one part was complementary to target H1N1 DNA and the other part was complementary to the signal DNA. In the absence of target H1N1 DNA, the G-rich fragment of QBF-DNA can form G-quadruplex-NMM complex, which outputted a fluorescent signal. With the presence of target H1N1 DNA, QBF-DNA, and A-DNA can simultaneously hybridize with target H1N1 DNA to form double-helix structure. In this case, the A-DNA partially hybridized with the QBF-DNA, which inhibited the formation of G-quadruplex-NMM complex, leading to the decrease of fluorescent signal. Under the optimum conditions, the fluorescence intensity was inversely proportional to the concentration of target H1N1 DNA over the range from 25 to 700 pmol/L with a detection limit of 8 pmol/L. In addition, the method is target specific and practicability, and would become a new diagnostic assay for H1N1 DNA sequence and other infectious diseases.

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.

We report the fabrication and characterization of a miniaturized electrochemical platform for the label-free detection of DNA hybridization. The proposed platform is fabricated using microfabrication and electrodeposition techniques. Comprising a Ti working electrode with electrodeposited Au nanostructures, and Pt/Au pseudo-reference and counter electrodes, the device accounts for a limit of detection of 0.97 fM and a sensitivity of 20.78 (μA μM-1) cm-2 with respect to Dengue virus specific consensus primer detection in the range of 10 fM-1 μM. Here, the incorporation of nanostructured Au in the active sensing area not only enhances the current response by increasing the overall surface area, but it also facilitates facile probe DNA immobilization by gold-thiol self-assembly. We have used differential pulse voltammetry analysis in this study to monitor the changes in reaction kinetics with respect to target hybridization. Furthermore, the evaluation of reproducibility of the biosensor and its selectivity against interference has yielded acceptable outcomes. Additionally, in order to evaluate the system's selectivity, we have successfully distinguished PCR amplified wild type and mutant target DNAs corresponding to the BRCA1 specific gene. Here, the mutant and the wild type target DNAs differ by a two base deletion, and the fact that the system is able to differentiate even such minute dissimilarities under hybridization conditions is indicative of its superior performance.

In the interest of developing novel electrocatalyst for high performance DNA biosensing, with distinctive chiral double helix nanostructure, nitrogen doped chiral carbonaceous nanotube (Chiral-CNT) was employed for ultrasensitive label-free DNA biosensing research. Chiral-CNT can quantitative detection of four DNA bases with high sensitivity and selectivity. Without any prehydrolysis and labeling process, direct electrochemistry of single-stranded DNA and double-stranded DNA, qualitative and quantitative detection of DNA hybridization (low detection limit: 0.0268 g L−1) were realized. Moreover, sensitive detection of DNA damage induced by fenton reagent was also realized with low detection limit of 0.0350 mg mL−1 and high sensitivity of 7.42 μA mg−1 mL. The high biosensing performance attributes to the unique chiral structure of Chiral-CNT, leads to efficient interreaction between Chiral-CNT and DNA molecule.

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

Electrochemical biosensors have extremely robust applications while offering ease of preparation, miniaturization, and tunability. By adjusting the arrangement and properties of immobilized probes on the sensor surface to optimize target–probe association, one can design highly sensitive and efficient sensors. In electrochemical nucleic acid biosensors, a self-assembled monolayer (SAM) is widely used as a tunable surface with inserted DNA or RNA probes to detect target sequences. The effects of inhomogeneous probe distribution across surfaces are difficult to study experimentally due to inadequate resolution. Regions of high probe density may inhibit hybridization with targets, and the magnitude of the effect may vary depending on the hybridization mechanism on a given surface. Another fundamental question concerns diffusion and hybridization of DNA taking place on surfaces and whether it speeds up or hinders molecular recognition. We used all-atom Brownian dynamics simulations to help answer these questions by simulating the hybridization process of single-stranded DNA (ssDNA) targets with a ssDNA probe on polar, nonpolar, and anionic SAMs at three different probe surface densities. Moreover, we simulated three tightly packed probe clusters by modeling clusters with different interprobe spacing on two different surfaces. Our results indicate that hybridization efficiency depends strongly on finding a balance that allows attractive forces to steer target DNA toward probes without anchoring it to the surface. Furthermore, we found that the hybridization rate becomes severely hindered when interprobe spacing is less than or equal to the target DNA length, proving the need for a careful design to both enhance target–probe association and avoid steric hindrance. We developed a general kinetic model to predict hybridization times and found that it works accurately for typical probe densities. These findings elucidate basic features of nanoscale biosensors, which can aid in rational design efforts and help explain trends in experimental hybridization rates at different probe densities.

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

Non-small cell lung cancer (NSCLC) accounts for ∼80-85% of all lung cancer cases, and the EML4-ALK fusion oncogene is a well-known contributor to NSCLC cases. Expensive methods such as FISH, IHC, and NGS have been used to detect the EML4-ALK fusion oncogene. Here, a cost-effective and facile method of detecting and differentiating an EML4-ALK fusion oncogene from the wild-type gene has been accomplished by DNA hybridization using the microfluidic biochip. First, oligonucleotide probes were confirmed for successful detection of immobilized sense strands. Second, capture of the sense PCR product strands (fusion and WT) and their subsequent detection and differentiation were accomplished. Our proof-of-concept study shows the ability to detect 1% fusion products, among WT ones.

An electrochemical DNA biosensor was designed for the detection of a specific target DNA after hybridization with a complementary DNA probe immobilized onto a glassy carbon electrode surface. Quercetin was successfully used as a new electroactive indicator for the hybridization detection. Different interactions of quercetin with single-stranded DNA (ss-DNA) and double-stranded DNA (ds-DNA) led to different electrochemical signals, which were recorded as cyclic and differential pulse voltammograms enabling hybridization detection. Various parameters influencing the biosensor performance were evaluated, and optimized conditions were obtained. Also, the detection limit of 83 pM with a relative standard deviation of 4.6% was obtained for the determination of complementary oligonucleotides. Then, the developed biosensor was applied successively for the detection of short sequences of hepatitis C virus (HCV1). The hybridization between the probe (PHCV1) and its complementary sequence (HCV1a) as the target was studied. Some hybridization experiments with noncomplementary oligonucleotides also showed that the suggested DNA sensor responds selectively to the target.

In the last decades, DNA has been considered far more than the system carrying the essential genetic instructions. Indeed, because of the remarkable properties of the base-pairing specificity and thermoreversibility of the interactions, DNA plays a central role in the design of innovative architectures at the nanoscale. Here, combining complementary DNA strands with a custom-made solution of silver nanoparticles, we realize plasmonic aggregates to exploit the sensitivity of Surface Enhanced Raman Spectroscopy (SERS) for the identification/detection of the distinctive features of DNA hybridization, both in solution and on dried samples. Moreover, SERS allows monitoring the DNA aggregation process by following the temperature variation of a specific spectroscopic marker associated with the Watson-Crick hydrogen bond formation. This temperature-dependent behavior enables us to precisely reconstruct the melting profile of the selected DNA sequences by spectroscopic measurements only.

Deoxyribonucleic acid (DNA) hybridization is at the heart of countless biological and biotechnological processes. Its theoretical modeling played a crucial role, since it has enabled extracting the relevant thermodynamic parameters from systematic measurements of DNA melting curves. In this article, we propose a framework based on statistical physics to describe DNA hybridization and melting in an arbitrary mixture of DNA strands. In particular, we are able to analytically derive closed expressions of the system partition functions for any number $N$ of strings and explicitly calculate them in two paradigmatic situations: (i) a system made of self-complementary sequences and (ii) a system comprising two mutually complementary sequences. We derive the melting curve in the thermodynamic limit ($N\ensuremath{\rightarrow}\ensuremath{\infty}$) of our description, which provides a full justification for the extra entropic contribution that in classic hybridization modeling was required to correctly describe within the same framework the melting of sequences either self-complementary or not. We thus provide a thorough study comprising limit cases and alternative approaches showing how our framework can give a comprehensive view of hybridization and melting phenomena.

Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.

Room-temperature plasma treated graphene based FET was firstly proposed for the DNA hybridization detection. Affinity and electrical properties of the graphene based DNA-FET sensor were studied and improved benefits from the surface modification. The facile room-temperature Ar plasma easily removes residues from the graphene surface and changes the hydrophilic properties of graphene, which is important for our solution gated DNA-FET sensor. Limit of the detection of below 10 aM is obtained in our experiment. Especially, DNA concentration (CDNA)/the amount of net drain current (ΔI) and the negative shift in the VCNP value of the GFET sensor with the plasma treated 30 s are all improved compared with that without treatment. It shows that the easily plasma treatment of the graphene surface can be used for the solution gated FET sensor.

Immunohistochemistry (IHC) can provide detailed information about protein expression within the cell microenvironment and is one of the most common techniques in biology and medicine due to the broad availability of highly specific antibodies and well-established bioconjugation methods for modification of these antibodies with chromogens and fluorophores. Despite recent advances in this field, it remains challenging to simultaneously achieve high multiplexing, sensitivity, and throughput in single-cell profiling experiments. Here, the combination of two powerful technologies is reported, quantum dot and signal amplification by exchange reaction (QD-SABER), for sensitive and multiplexed imaging of endogenous proteins. Compared to the conventional IHC process using dye-labeled secondary antibodies (which already has a built-in signal amplification mechanism), QD-SABER provides an additional 7.6-fold signal amplification. In addition, the DNA hybridization-based IHC can be rapidly removed to regenerate the sample for subsequent cycles of immunostaining (>10 cycles), greatly expanding the multiplexing capability.

Early screening of L. monocytogenes in ready-to-eat food can prevent and control its harmful effects. In this study, we propose a highly sensitive magnetic DNA sensor based on nucleic acid hybridization reaction and magnetic signal readout. We design the L. monocytogenes specific probe1 and probe2 and label them on the 30 and 250 nm magnetic nanoparticles, respectively. The hybridization reaction between the magnetic probes and DNA of L. monocytogenes could form a sandwich nanocomplex. After magnetic separation, the unbound MNP30–probe2 can act as the transverse relaxation time (T2) signal readout probe. This assay allows the one-step detection of L. monocytogenes as low as 50 CFU/mL within 2 h without DNA amplification, and the average recovery in the spiked ham sausage samples can reach 92.6%. This system integrates the high sensitivity of magnetic sensing and high efficiency of hybridization reaction, providing a promising detection platform for pathogens.

Nanometer-sized liposomes decorated with macromolecules are increasingly used as drug delivery vehicles due to their long lifetimes and target cell specificity, but surface characterization methods often change their properties, which leads to incorrect results. Ligand binding is commonly applied for characterizing these surface modifications. Here, we use a nanofluidic-based label-free sensor for real-time sensing of ligands binding to liposomes. The liposomes are trapped in a nanochannel with a salt concentration gradient, and as the trapping position depends on the liposomes' zeta potential, it changes when charged ligands bind to the liposomes. Our sensing method does not require immobilization of the liposomes or labeling of the ligands with fluorophores, which may both affect the sensing. The zeta potential sensing is demonstrated by measuring hybridization of DNA targets with complementary DNA probes on liposome surfaces. DNA hybridization is monitored for both ensembles and individual liposomes, the latter allows for analysis of ensemble heterogeneity, and we demonstrate sensitivity to changes in surface charge down to 1.5%. DNA hybridization is used to demonstrate label-free sensing, but the method also has potential applications within exosome characterization, where biorecognition of, e.g., surface DNA, proteins, and antibodies is a promising candidate for early stage cancer diagnostics.

40 páginas, 3 tabla, 1 figura, 3 figuras suplementarias. -- The definitive version is available at http://www.elsevier.com, A polyphasic study was undertaken to establish the taxonomic status of three Modestobacter strains isolated from a high altitude Atacama Desert soil. The isolates, strains 1G6T, 1G14 and 1G50, showed chemotaxonomic and morphological properties characteristic of members of the genus Modestobacter. The peptidoglycan contained meso-diaminopimelic acid, the whole cell sugars were glucose and ribose (diagnostic sugars) and arabinose, the predominant menaquinone was MK-9(H4), polar lipid patterns contained diphosphatidylglycerol, glycophosphatidylinositol, phosphatidylethanolamine (diagnostic component), phosphatidylglycerol and phosphatidylinositol while whole cellular fatty acid profiles consisted of complex mixtures of saturated, unsaturated iso- and anteiso-components. The isolates were shown to have different BOX-PCR fingerprint and physiological profiles. They formed a distinct phyletic line in Modestobacter 16S rRNA gene trees, were most closely related to the type strain of Modestobacter italicus (99.9 % similarity) but were distinguished from this and other closely related Modestobacter type strains using a combination of phenotypic properties. Average nucleotide identity and digital DNA:DNA hybridization similarities between the draft genome sequences of isolate 1G6T and M. italicus BC 501T were 90.9 % and 42.3 %, respectively, indicating that they belong to different species. Based on these phenotypic and genotypic data it is proposed that the isolates be assigned to a novel species in the genus Modestobacter, namely as Modestobacter excelsi with isolate 1G6T (=DSM 107535T =PCM 3004T) as the type strain. Analysis of the whole genome sequence of M. excelsi 1G6T (genome size of 5.26 Mb) showed the presence of genes and gene clusters that encode for properties that are in tune with its adaptation to extreme environmental conditions that prevail in the Atacama Desert biome., This work was supported by Grant No. 2017/01/X/NZ8/00140 from the National Science Centre (NCN) of Poland. Genome sequencing was provided by MicrobesNG (https://www.microbesng.uk) which is supported by the BBSRC (grant number BB/L024209/1).

It remains a technical challenge to accurately identify close species of herbal medicines, especially from adulterants, because of their highly identical phenotypes and chemical compositions. Here, we report a direct, sequencing-free, high-curvature nanostructuring-based electrochemical herb sensor (nanoE-herb sensor) to identify herbal species quickly and accurately using ITS2 barcodes. We engineer a nano-roughened carbon-supported gold nanostructuring array by photolithograph-free, one-step electrodeposition. The 3D fractal nanostructures exhibit a high deflection angle that largely enhances DNA hybridization efficiency, particularly for the midcomplementary hybridization, as compared to the 2D planar surface. More importantly, such a trans-scale array biointerface (including macroscale carbon and nanoscale gold branches) can overcome the detection barrier of slow diffusion of a long genomic sequence and inaccessibility of the sequestered variations in ITS2 secondary structures through the out-protruded 3D functional nanostructures. Our nanoE-herb sensor achieves a detection limit of 0.18 fM for the 64-mer fragment of saffron ITS2 barcode with midhybridization and shows superior specificity against even single-base mismatch. The sensor also precisely differentiates saffron from six other adulterants by directly detecting unpurified asymmetric PCR amplicons (∼500 bp) with ITS2 sequences, suggesting its great potential in the field identification of herbal medicinal species and pathogenic bacteria with specific DNA barcodes.