A Competitor-switched Electrochemical Sensor for Detection of DNA

A competitor‐switched electrochemical sensor based on a generic displacement strategy was designed for DNA detection. In this strategy, an unmodified single‐stranded DNA (cDNA) completely complementary to the target DNA served as the molecular recognition element, while a hairpin DNA (hDNA) labeled with a ferrocene (Fc) and a thiol group at its terminals served as both the competitor element and the probe. This electrochemical sensor was fabricated by self‐assembling a dsDNA onto a gold electrode surface. The dsDNA was pre‐formed through the hybridization of Fc‐labeled hDNA and cDNA with their part complementary sequences. Initially, the labeled ferrocene in the dsDNA was far from surface of the electrode, the electrochemical sensor exhibited a "switch‐off" mode due to unfavorable electron transfer of Fc label. However, in the presence of target DNA, cDNA was released from hDNA by target DNA, the hairpin‐open hDNA restored its original hairpin structure and the ferrocene approached onto the electrode surface, thus the electrochemical sensor exhibited a "switch‐on" mode accompanying with a change in the current response. The experimental results showed that as low as 4.4×10⁻¹⁰ mol/L target DNA could be distinguishingly detected, and this method had obvious advantages such as facile operation, low cost and reagentless procedure.     

Role of loop entropy in the force induced melting of DNA hairpin

Dynamics of a single stranded DNA, which can form a hairpin have been studied in the constant force ensemble. Using Langevin dynamics simulations, we obtained the force-temperature diagram, which differs from the theoretical prediction based on the lattice model. Probability analysis of the extreme bases of the stem revealed that at high temperature, the hairpin to coil transition is entropy dominated and the loop contributes significantly in its opening. However, at low temperature, the transition is force driven and the hairpin opens from the stem side. It is shown that the elastic energy plays a crucial role at high force. As a result, the force-temperature diagram differs significantly with the theoretical prediction.     

Hairpin Assembly Amplified Electrochemical Biosensor for Highly Sensitive and Specific Detection of DNA

In this report, a simple electrochemical biosensor has been developed for highly sensitive and specific detection of DNA based on hairpin assembly amplification. In the presence of target DNA, the biotin‐labelled hairpin H1 is opened by hybridizing with target DNA through complementary sequences. Then the opened hairpin H1 assembles with the hairpin H2 to displace the target DNA, generating H1‐H2 complex. The displaced target DNA could trigger the next cycle of hairpins assembly, resulting in the generation of numerous H1‐H2 complexes. Subsequently, the H1‐H2 complex hybridizes with the capture probe immobilized on the electrode. Finally, the streptavidin alkaline phosphatase (ST‐ALP) binds to biotin in the capture probe‐H1‐H2 complex and catalyzes the substrate α‐naphthol (α‐NP) to produce electrochemical signal. To make a more fascinating hairpin assembly amplification strategy in signal amplification, mismatched base sequences are designed in hairpin H2 to decrease non‐specific binding of the hairpin substrates. The developed biosensor achieves a sensitivity of 20 pM with a linear range from 25 pM to 25 nM, and shows high selectivity toward single‐base mismatch. Thus, the proposed electrochemical biosensor might have the potential for early clinical diagnosis and therapy.     

Catalytic Hairpin Assembly‐Programmed DNA Three‐Way Junction for Enzyme‐Free and Amplified Electrochemical Detection of Target DNA

DNA three‐way junctions (DNA 3WJ) have been widely used as important building blocks for the construction of DNA architectures and dynamic assemblies. Herein, we describe for the first time a catalytic hairpin assembly‐programmed DNA three‐way junction (CHA‐3WJ) strategy for the enzyme‐free and amplified electrochemical detection of target DNA. It takes full advantage of the target‐catalyzed hairpin assembly‐induced proximity effect of toehold and branch‐migration domains for the ingenious execution of the strand displacement reaction to form the DNA 3WJ on the electrode surface. A low detection limit of 0.5 pM with an excellent selectivity was achieved for target DNA detection. The developed CHA‐3WJ strategy also offers distinct advantages of simplicity in probe design and biosensor fabrication, as well as enzyme‐free operation. Thus, it opens a promising avenue for applications in bioanalysis, design of DNA‐responsive devices, and dynamic DNA assemblies.     

Photoactivated DNA Assembly and Disassembly for On-Demand Activation and Termination of cGAS-STING Signaling

Despite significant progress in DNA self-assembly for interfacing with biology, spatiotemporally controlled regulation of biological process via in situ dynamic DNA assembly remains an outstanding challenge. Here, we report an optically triggered DNA assembly and disassembly strategy that enables on-demand activation and termination of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. In the design, an activatable DNA hairpin is engineered with a photocleavable group at defined site to modulate its self-assembly activity. Light activation induces the configurational switching and consequent self-assembly of the DNA hairpins to form long linear double-stranded structures, allowing to stimulate cGAS protein to synthesize 2′,3′-cyclic-GMP-AMP (cGAMP) for STING stimulation. Furthermore, by endowing the pre-assembled DNA scaffold with a built-in photolysis feature, we demonstrate that the cGAS-STING stimulation can be efficiently terminated through remote photo-triggering, providing for the first time a route to control the temporal “dose” on-demand for such a stimulation. We envision that this regulation strategy will benefit and inspire both fundamental research and therapeutic applications regarding the cGAS-STING pathway.     

Protein-binding molecular switches via host-guest stabilized DNA hairpins

Molecular switches, with target protein-binding activity controlled by prior binding to specific input stimuli, are ubiquitously used in Nature. However, the emulation of such responsive systems, especially in a de novo fashion, remains a significant challenge. Herein, we disclose a strategy that harnesses an intramolecular β-CD/adamantane host-guest interaction to generate a stabilized DNA hairpin (ΔT(m) = 17 °C) that undergoes an input oligonucleotide (ODN)-selective structural transformation from a stem-loop conformation to a duplex. This ODN-induced conformational switch allows for the transition from an inactive state (wherein the adamantane protein-binding headgroup is encapsulated) to an activated protein-binding complex, with a freely accessible adamantane moiety. Given that hairpin domains can be readily modulated to be responsive to alternative ODN triggering sequences and that encapsulating macrocycles, such as β-CD, are good hosts for a number of protein-binding small molecules, this strategy may furnish a general method to develop ODN-responsive protein-binders.     

Molecular assessment of S1 endonuclease-resistant snapback hairpin loops generated by DNA polymerase I during the in-vitro nick translation reaction

The in-vitro nick translation reaction used to label DNA to high specific activity also produces aberrant DNA structures known as “snapback” hairpin loops. Hairpin structures are precluded from participating in precise DNA-DNA hybridization interactions. Three nick translation systems were all found to yield significant quantities of snapback hairpins, as determined by their resistance to S1 endonuclease digestion following denaturation. The relative quantities of hairpins produced correlated with both the mass average size of the final DNA probe product synthesized as well as the overall rate of the nick translation reaction. Decreases in the amount of exogenous DNase I used in nick translation reactions produced significant decreases in the amount of hairpin loop structures formed. Hairpins could be effectively removed from nick-translated DNAs by employing hydroxylapatite column chromatography. Strategies to reduce hairpin formation during nick translation and the removal of hairpins from nick-translated DNAs are presented.     

A thermostable azo-linker for reversible photoregulation of DNA replication

Small molecules such as azobenzenes, one of the best reversible photo-switches, can be covalently incorporated into DNA to regulate its structures and functions with irradiation of the specific wavelengths. Using this strategy, a thermostable azobenzene linker was employed to construct modified oligodeoxynucleotides, and we successfully achieve reversible photoregulation of DNA replication in vitro with short irradiation time. Five minutes UV irradiation for regulating trans→cis transformation can minimize DNA damage, still ensure the polymerase reaction of cis-form. Formation of DNA hairpin structure can also be controlled by photoregulation using this linker.     

Amplified electrochemical DNA sensor using peroxidase-like DNAzyme

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H₂O₂, which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.     

Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events

The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but also this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.     

Long-distance radical cation transport in DNA: Horizontal charge hopping in a dimeric quadruplex

A series of DNA hairpins were synthesized and shown to associate to form quadruplexes formed by stacking five G-quartets in an antiparallel orientation. One of the hairpins in the quadruplex was linked covalently at the 5'-end to an anthraquinone (AQ) group and a 32P label was incorporated either at the 3'-terminus of the AQ-containing hairpin or on its partner hairpin in the quadruplex. Irradiation of the AQ group with UV light leads to the one-electron oxidation of the DNA and concomitant introduction of a radical cation into the DNA. Analysis by PAGE and autoradiography shows that the radical cation reacts at guanines both on the AQ-containing strand and with its partner hairpin in the quadruplex. This observation demonstrates that charge migration in DNA occurs vertically along a DNA chain and horizontally within a G-quartet.     

Immobilization of DNA on magnetic microparticles for mercury enrichment and detection with flow cytometry

Mercury detection in water has attracted a lot of research interest due to its highly toxic nature and adverse environmental impact. In particular, the recent discovery of specific binding of Hg(II) to thymine-rich (T-rich) DNA resulting in T-Hg(II)-T base pairs has led to the development of a number of sensors with different signaling mechanisms. However, the majority of such sensors were non-immobilized. Immobilization, on the other hand, allows active mercury adsorption, signal amplification, and sensor regeneration. In this work, we immobilized a thymine-rich DNA on a magnetic microparticle (MMP) surface through biotin-streptavidin interactions. In the presence of Hg(II), the DNA changes from a random coil structure into a hairpin, upon which SYBR Green I binds to emit green fluorescence. Detection was carried out by using flow cytometry where the fluorescence intensity increased ≈9-fold in the presence of mercury and the binding of mercury reached equilibrium in less than 2 min. The sensor showed a unique sample-volume-dependent fluorescence signal change where a higher fluorescence was obtained with a larger sample volume, suggesting that the particles can actively adsorb Hg(II). Detection limits of 5 nM (1 ppb) and 14 nM (2.8 ppb) were achieved in pure buffer and in mercury-spiked Lake Ontario water samples, respectively.     

Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.     

Probing the complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy

The conformational fluctuations of dye-quencher labeled DNA hairpin molecules in aqueous solution were investigated using dual probe beam fluorescence fluctuation spectroscopy. The measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset optical probe regions, the absolute and relative concentrations of each conformational substate of the DNA, and the kinetics of the DNA hairpin folding and unfolding reactions in the 1 micros to 10 ms time range. A DNA hairpin containing a 21-nucleotide polythymine loop and a 4-base pair stem exhibited double exponential relaxation kinetics, with time constants of 84 and 393 micros. This confirms that folding and melting of the DNA hairpin structure is not a two state process but proceeds by way of metastable intermediate states. The fast time constant corresponds to formation and unfolding of an intermediate, and the slow time constant is due to formation and disruption of the fully base-paired stem. This is consistent with a previous study of a similar DNA hairpin with a 5-base pair stem, in which the fast reaction was attributed to the fluctuations of an intermediate DNA conformation [J. Am. Chem. Soc. 2006, 128, 1240-1249]. In that case, reactions involving the native conformation could not be observed directly due to the limited observation time range of the fluorescence correlation spectroscopy experiment. The intermediate states of the DNA hairpins are suggested to be due to a collapsed ensemble of folded hairpins containing various partially folded or misfolded conformations.     

Ultrasensitive Electrochemical Biosensor for HPV16 Oncogene Based on Y-shaped DNA Catalytic Hairpin Assembly and Template-free DNA Extension Reaction

An ultrasensitive electrochemical biosensor for HPV16 oncogene was explored. Hairpin DNA-1, which can specifically bind with HPV16 oncogene, was fixed on the surface of gold electrode. Two hairpin DNAs underwent catalytic hairpin assembling with hairpin DNA-1 to construct Y-shaped DNA nanostructure, liberating HPV16 oncogene for target recycling. The 3’ terminus of Y-shaped DNA nanostructure was prolonged under the catalysis of terminal deoxynucleotidyl transferase. Methylene blue was adsorbed onto DNA nanostructure to generate characteristic differential pulse voltammetry signal. This signal was increased with the concentration of HPV16 oncogene, and the detection limit of HPV16 oncogene was as low as 0.19 fM.     

A novel DNA hairpin substrate for bleomycin

A 16-nucleotide DNA hairpin containing 4-aminobenzo[g]quinazoline-2-one 2'-deoxyribose at position 15 has been prepared and found to lack significant fluorescence. When treated with Fe(II).BLM, the hairpin was found to undergo oxidative transformation selectively at position 15. The predominant fluorescent product was characterized and quantified. The pro-fluorescent DNA hairpin was used as a substrate for 15 bleomycin congeners, and the results were compared with those obtained following cleavage of a radiolabeled DNA duplex and PAGE analysis.     

Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

Electrospray ionization mass spectrometric analysis of noncovalent complexes between DNA and polyamides containing N-methylpyrrole and N-methylimidazole

Electrospray ionization mass spectrometry was utilized to investigate the noncovalent complexes between novel polyamides and DNA containing the TCCT sequence. We analyzed the noncovalent binding of the polyamides with the DNA and assessed their relative affinities and stoichiometry. The results confirm that hairpin polyamides have higher binding affinities than three-ring polyamides. The hairpin polyamide (PyPyPyPygammaPyImImPybetaDp) has the highest affinity, and the beta-linked polyamide (PyPyPybetaImImImbetaDp) shows a dominant 1:2 binding stoichiometry. Two groups of competition experiments were undertaken to compare the binding affinities of the duplex DNA with different polyamides directly. The affinity scale thus obtained for the group-1 polyamides is PyPyPyPygammaPyImImPybetaDp > PyPyPybetaImImImbetaDp approximately PyPyPygammaImImImbetaDp > PyPyPybetaDp > PyImImbetaDp approximately ImImPybetaDp, and the order for the group-2 polyamides is PyPyPygammaImImImbetaDp > PyPyPygammaImImImbetaOEt > PyPyPygammaImImImbetaCOOH.