DNA Conformational Changes Research Articles

Adamantane Derivatives Functionalized Gold Nanoparticles for Colorimetric Detection of MiRNA

MiRNAs are riveting RNA molecules due to their close relevance to the regulation of gene expression and certain physiological or pathological processes. Rapid and sensitive methods for miRNA assay are essential for biological researches and clinical diagnosis of many diseases. In this work, gold nanoparticles (AuNPs) have been modified with adamantane derivatives and then Dwith NA probes for colorimetric detection of miRNA. Target miRNA induces a change in DNA conformation and initiates strand displacement amplification, eventually leading to massive aggregations of AuNPs. By comparing the UV–vis absorption spectra, miRNA concentration can be determined. This developed method can detect miRNA as low as 3.7 × 10−15m with remarkable specificity. Moreover, it is successfully used to inspect the expression of miRNA in biological samples. Therefore, adamantane derivatives functionalized AuNPs are demonstrated to offer a novel platform for biosensing and the miRNA sensing strategy may find a broad spectrum of practical applications.

A network of cis and trans interactions is required for ParB spreading.

Most bacteria utilize the highly conserved parABS partitioning system in plasmid and chromosome segregation. This system depends on a DNA-binding protein ParB, which binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA over multiple kilobases in a phenomenon called spreading. Previous single-molecule experiments in combination with genetic, biochemical and computational studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystallized with parS revealed that ParB dimers tetramerize to form a higher order nucleoprotein complex. Using this structure as a guide, we systematically ablated a series of proposed intermolecular interactions in the Bacillus subtilis ParB (BsSpo0J) and characterized their effect on spreading using both in vivo and in vitro assays. In particular, we measured DNA compaction mediated by BsSpo0J using a recently developed single-molecule method to simultaneously visualize protein binding on single DNA molecules and changes in DNA conformation without protein labeling. Our results indicate that residues acting as hubs for multiple interactions frequently led to the most severe spreading defects when mutated, and that a network of both cis and trans interactions between ParB dimers is necessary for spreading.

Label-Free Homogeneous Electroanalytical Platform for Pesticide Detection Based on Acetylcholinesterase-Mediated DNA Conformational Switch Integrated with Rolling Circle Amplification

This study addresses the need for sensitive pesticide assay by reporting a new label-free and immobilization-free homogeneous electroanalytical strategy, which combines acetylcholinesterase (AChE)-catalyzed hydrolysis product-mediated DNA conformational switch and rolling circle amplification (RCA) to detect organophosphorous and carbamate pesticides in a "signal-on" mode. When target pesticides were present, AChE activity was inhibited and could not trigger the following DNA conformational change and the RCA reaction, which results in numerous methylene blue (MB) molecules in a free state, generating a strong electrochemical response. This proposed strategy was highly sensitive for omethoate detection with a detection limit as low as 2.1 μg/L and a linear range from 10 to 10 000 μg/L. Furthermore, this strategy was demonstrated to be applicable for pesticide detection in real samples. Thus, this novel label-free homogeneous electroanalytical strategy holds great promise for pesticide detection and can be further exploited for sensing applications in the environment and the food safety field.

Silver(I)-Induced Conformation Change of DNA: Gold Nanocluster as a Spectroscopic Probe

DNA-based materials are promising avenues for designing optical sensors, light-harvesting devices, energy conversion devices, and other potential applications. Therefore, the understanding of DNA conformation is important for designing such materials. Here, we investigate the conformation of DNA by a photoexcited energy transfer process where the Au cluster is covalently attached with AlexaFluor 488 (A488) dye tagged DNA. Fluorescence resonance energy transfer (FRET) is a well-known method to determine the donor–acceptor distance where A488 acts as donor and the gold nanoclusters (Au NCs) act as acceptor. Spectroscopic studies reveal that Ag+ ions are incorporated into dsDNA to produce a Ag+-C (cytosine) metal base pair bond as the used DNA contains 55.6% G-C base pairs. Steady-state and time-resolved spectroscopic studies reveal that the distances between donor and acceptor are found to be 98 and 83 Å in the absence and presence of Ag+ ion, respectively, indicating the rigid conformation of dsDNA becomes flexible in the presence of a Ag+ ion. The efficiency of energy transfer increases from 63% to 82% in the presence of the Ag+ ion because the conformation of DNA changes with the formation of DNA-metal base pairs. Analysis suggests that the Au cluster is a useful spectroscopic probe to understand the conformation of DNA and others. Furthermore, this energy transfer from dye to Au NCs using the DNA scaffold may open up new opportunities in designing an artificial light-harvesting system.

Condensin, master organizer of the genome.

A fundamental requirement in nature is for a cell to correctly package and divide its replicated genome. Condensin is a mechanical multisubunit complex critical to this process. Condensin uses ATP to power conformational changes in DNA to enable to correct DNA compaction, organization, and segregation of DNA from the simplest bacteria to humans. The highly conserved nature of the condensin complex and the structural similarities it shares with the related cohesin complex have provided important clues as to how it functions in cells. The fundamental requirement for condensin in mitosis and meiosis is well established, yet the precise mechanism of action is still an open question. Mutation or removal of condensin subunits across a range of species disrupts orderly chromosome condensation leading to errors in chromosome segregation and likely death of the cell. There are divergences in function across species for condensin. Once considered to function solely in mitosis and meiosis, an accumulating body of evidence suggests that condensin has key roles in also regulating the interphase genome. This review will examine how condensin organizes our genomes, explain where and how it binds the genome at a mechanical level, and highlight controversies and future directions as the complex continues to fascinate and baffle biologists.

DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase.

8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O8 and H7). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine-DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition. Here, we use molecular dynamics simulations to explore the mechanism by which OGG1 discriminates between 8-oxoG and guanine along the base-eversion pathway. The MD results suggest an important role for kinking of the DNA by the glycosylase, which positions DNA phosphates in a way that assists lesion recognition during base eversion. The computational predictions were validated through experimental enzyme assays on phosphorothioate substrate analogs. Our simulations suggest that OGG1 distinguishes between 8-oxoG and G using their chemical dissimilarities not only at the active site but also at earlier stages during base eversion, and this mechanism is at least partially conserved in Fpg despite a lack of structural homology. The similarity also suggests that lesion recognition through multiple gating steps may be a common theme in DNA repair. Our results provide new insight into how enzymes can exploit kinetics and DNA conformational changes to probe the chemical modifications present in DNA lesions.

Asymmetric DNA Unwrapping Drives Sequential Dimer Release in Nucleosomes

The fundamental structures of genome packaging in the nucleus are nucleosomes, whose stability and dynamics provide key mechanisms for the epigenetic control of genes. The canonical nucleosome consists of 147 basepairs of DNA tightly wrapped around an octamer of histone proteins. In vitro and in vivo studies reveal a growing diversity of nucleosomal structures with varying DNA conformations and histone compositions that facilitate epigenetic activity. Since characterization of the kinetic pathways between different biologically relevant nucleosomes species remain elusive, we developed a novel biophysical method to probe the coordination between changes in DNA conformation and reconfiguration of the histone core.

Probing Manganese Ion Distributions Around Nucleic Acids using Small Angle X-Ray Scattering

Manganese ions are divalent cations that have profound biological roles in cellular processes, e.g. they are involved in folding of some RNAs and in some DNA conformational changes. We use two experimental Small-Angle X-ray Scattering (SAXS) based approaches to probe the distribution of Manganese ions around nucleic acids. SAXS is a solution-based technique that is sensitive to the ion cloud and hydration layer around nucleic acids. By absolutely calibrating SAXS data, we can accurately characterize the ion cloud distribution and the water layer around DNA.

Monitoring the Intercalation of Acridine Orange (AO) Molecules into 6-Methyl isoxanthopterin (6-MI)-Labeled DNA by Circular Dichroism and Single-Molecule FRET

Intercalation into duplex DNA occurs when a planar and aromatic small molecule ligand binds between adjacent base pairs of duplex DNA, forming a sandwich-like structure. This insertion significantly perturbs the double-stranded DNA structure. As a result, such intercalation may also perturb DNA-structure-dependent biological processes, such as DNA transcription, replication and repair. Based on such functional perturbations, extensive studies have been performed that focus on the development of intercalative drugs with possible clinical potential. Among the intercalators that have been examined, acridine derivatives have been much investigated. However, more work is required to develop effective drugs with the desired selectivities and reduced side effects. In an attempt to provide more incisive molecular approaches to understanding intercalation mechanisms, we have shown that 6-methyl isoxanthopterin (6-MI) and acridine orange (AO) can act as a donor-acceptor Forster Resonance Energy Transfer (FRET) pair. 6-MI is a fluorescent guanine analogue that can serve as a local reporter of DNA conformational changes, while acridine orange is a well-studied DNA intercalator. We show how these two optical probes can be paired to monitor local AO-DNA intercalation dynamics with multi-millisecond time resolution. Applying single-molecule FRET and linear dichroism methods developed in our group, we are using these approaches to study the roles of intermediate conformational states during the intercalation process.

Enhancement of magnetofection efficiency using chitosan coated superparamagnetic iron oxide nanoparticles and calf thymus DNA

Superparamagnetic iron oxide nanoparticles (MNPs) were prepared and coated with chitosan (CS). The chitosan-magnetic iron oxide nanoparticles (CS-MNPs) were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), and the morphology of the particles was studied by transmission electron microscopy (TEM). Our findings show that the magnetic particles were monodisperse (10nm mean diameter) and exhibited superparamagnetic behavior. The interaction between the particles and calf-thymus DNA (DNA) in physiological buffer was studied with UV–vis, fluorescence and circular dichroism spectroscopy and zeta potential. Spectroscopic studies were indicated DNA conformational changes in the presence of CS-MNPs. Binding and thermodynamic parameters at different temperatures were calculated using the Stern–Volmer, Hill, Scatchard and Van’t Hoff equations. The binding process was spontaneous and interactions were electrostatic with the appropriate binding constant (Kb=4.52×103M−1, 3.69×103M−1 and 3.02×103M−1 at 300K, 310K and 320K, respectively). Zeta potential measurements of DNA continually increased with the addition of CS-MNPs, supporting our thermodynamic findings. Moreover, CS-MNPs were able to quench the fluorescence of DNA-intercalated ethidium bromide (DNA-EB) by a static quenching mechanism. Cytotoxicity studies show that the DNA-CS-MNP system is biocompatible with a human foreskin fibroblast cell line, HFFF2. Collectively, these results suggest that surface cationic magnetic chitosan-iron oxide nanoparticles can potentially enhance magnetofection efficiency.

Specific Conformational Change in Giant DNA Caused by Anticancer Tetrazolato-Bridged Dinuclear Platinum(II) Complexes: Middle-Length Alkyl Substituents Exhibit Minimum Effect.

Derivatives of the highly antitumor-active compound [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N2,N3)]2+ (5-H-Y), which is a tetrazolato-bridged dinuclear platinum(II) complex, were prepared by substituting a linear alkyl chain moiety at C5 of the tetrazolate ring. The general formula for the derivatives is [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)]2+, where R is (CH2)nCH3 and n = 0 to 8 (complexes 1-9). The cytotoxicity of complexes 1-4 in NCI-H460 human non-small-cell lung cancer cells decreased with increasing alkyl chain length, and those of complexes 5-9 increased with increasing alkyl chain length. That is, the in vitro cytotoxicity of complexes 1-9 was found to have a U-shaped association with alkyl chain length. This U-shaped association is attributable to the degree of intracellular accumulation. Although circular dichroism spectroscopic measurement indicated that complexes 1-9 induced comparable conformational changes in the secondary structure of DNA, the tetrazolato-bridged complexes induced different degrees of DNA compaction as revealed by a single DNA measurement with fluorescence microsopy, which also had a U-shaped association with alkyl chain length that matched the association observed for cytotoxicity. Complexes 7-9, which had alkyl chains long enough to confer surfactant-like properties to the complex, induced DNA compaction 20 or 1000 times more efficiently than 5-H-Y or spermidine. A single DNA measurement with transmission electron microscopy revealed that complex 8 formed large spherical self-assembled structures that induced DNA compaction with extremely high efficiency. This result suggests that these structures may play a role in the DNA compaction that was induced by the complexes with the longer alkyl chains. The derivatization with a linear alkyl chain produced a series of complexes with unique cellular accumulation and DNA conformational change profiles and a potentially useful means of developing next-generation platinum-based anticancer drugs. In addition, the markedly high ability of these complexes to induce DNA compaction and their high intracellular accumulation emphasized the difference in mechanism of action from platinum-based anticancer drugs.

An optimization algorithm for single-molecule fluorescence resonance (smFRET) data processing

The single-molecule fluorescence resonance energy transfer (smFRET) technique plays an important role in the development of biophysics. Measuring the changes of the fluorescence intensities of donor and acceptor and of the FRET efficiency can reveal the changes of distance between the labeling positions. The smFRET may be used to study conformational changes of DNA, proteins and other biomolecules. Traditional algorithm for smFRET data processing is highly dependent on manual operation, leading to high noise, low efficiency and low reliability of the outputs. In the present work, we propose an automatic and more accurate algorithm for smFRET data processing. It consists of three parts: algorithm for automatic pairing of donor and acceptor fluorescence spots based on negative correlation between their intensities; algorithm for data screening by eliminating invalid fluorescence spots sections; algorithm for global data fitting based on Baum-Welch algorithm of hidden Markov model (HMM). Based on the law of energy conservation, the light intensity of one pair of donor and acceptor shows a negative correlation. We can use this feature to find the active smFRET pairs automatically. The algorithm will first find out three active smFRET pairs with correlation coefficient lower than the threshold we set. This three active smFRET pairs will provide enough coordinate data for the algorithm to calculate the pairing matrix in the rest of automatic pairing work. After obtaining all the smFRET pairs, the algorithm for data screening will check the correlation coefficient for each pair. The invalid pairs with correlation coefficient higher than the threshold value will be eliminated. The rest of smFRET pairs will be analyzed by the data fitting algorithm. The Baum-Welch algorithm can be used for learning the global parameters. The global parameters we obtained will then be used to fit each FRET-time curve with Viterbi algorithm. The global parameter learning part will help us find the specific FRET efficiency for each state and the curve fitting part will provide more kinetic parameters. The optimization algorithm significantly simplifies the procedures of manual operation in the traditional algorithm and eliminate several types of noises from the experimental data automatically. We apply the new optimization algorithm to the analyses of folding kinetics data for human telomere repeat sequence, the G-quadruplex DNA. It is demonstrated that the optimization algorithm is more efficient to produce data with higher S/N ratio than the traditional algorithm. The final results reveal clearly the folding of G-quadruplex DNA in multiple states that are influenced by the K+ concentration.

Development of 19F-NMR chemical shift detection of DNA B-Z equilibrium using 19F-NMR.

Various DNA conformational changes are in correlation with biological events. In particular, DNA B-Z equilibrium showed a high correlation with translation and transcription. In this study, we developed a DNA probe containing 5-trifluoromethylcytidine or 5-trifluoromethylthymidine to detect DNA B-Z equilibrium using 19F-NMR. Its probe enabled the quantitative detection of B-, Z-, and ss-DNA based on 19F-NMR chemical shift change.

Interaction features of adenine DNA glycosylase MutY from E. coli with DNA substrates

Kinetic characteristics of specific recognition of damaged base by the DNA glycosylase MutY in model DNA substrates, containing oxoG/A-, G/A-, oxoG/C- and F/G pairs in the central position, were investigated. Conformational changes of the MutY enzyme during the recognition of the damaged base in DNA have been recorded by the change in the fluorescence intensity of tryptophan residues using the stopped-flow technique in real time. DNA duplexes containing a fluorescein residue were used for the registration of DNA conformational changes. Analysis of the kinetic curves allowed us to determine the values of rate constants for the kinetic stages of the interaction. It was shown that nonspecific contacts between the DNA-binding site of the enzyme and the DNA duplex are formed at the first stage of the interaction. It was found that the discrimination of Gua and oxoGua bases occurs at the second stage of the MutY interaction with the DNA duplex. The data obtained for the oxoG/C-substrate showed that the recognition of the base located opposite oxoGua also occurs at this stage.

Exercise Training and Epigenetic Regulation: Multilevel Modification and Regulation of Gene Expression.

Exercise training elicits acute and adaptive long term changes in human physiology that mediate the improvement of performance and health state. The responses are integrative and orchestrated by several mechanisms, as gene expression. Gene expression is essential to construct the adaptation of the biological system to exercise training, since there are molecular processes mediating oxidative and non-oxidative metabolism, angiogenesis, cardiac and skeletal myofiber hypertrophy, and other processes that leads to a greater physiological status. Epigenetic is the field that studies about gene expression changes heritable by meiosis and mitosis, by changes in chromatin and DNA conformation, but not in DNA sequence, that studies the regulation on gene expression that is independent of genotype. The field approaches mechanisms of DNA and chromatin conformational changes that inhibit or increase gene expression and determine tissue specific pattern. The three major studied epigenetic mechanisms are DNA methylation, Histone modification, and regulation of noncoding RNA-associated genes. This review elucidates these mechanisms, focusing on the relationship between them and their relationship with exercise training, physical performance and the enhancement of health status. On this chapter, we clarified the relationship of epigenetic modulations and their intimal relationship with acute and chronic effect of exercise training, concentrating our effort on skeletal muscle, heart and vascular responses, that are the most responsive systems against to exercise training and play crucial role on physical performance and improvement of health state.

Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470 nm) and methicillin-resistant Staphylococcus aureus

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibiotic resistant bacterium that leads to fatal infections; however, the mechanism of bacterial death remains unclear. In this paper, to uncover the mechanism underlying the bactericidal effect of blue light, a combination of Fourier transform infrared (FTIR) spectroscopy and chemometric tools is employed to detect the photoreactivity of MRSA and its distinctive pathway toward apoptosis after treatment. The mechanism of action of UV light and vancomycin against MRSA is also investigated to support the findings. Principal component analysis followed by linear discriminant analysis (PCA- LDA) is employed to reveal clustering of five groups of MRSA samples, namely untreated (control I), untreated and incubated at ambient air (control II), irradiated with 470nm blue light, irradiated with 253.5 UV light, and vancomycin-treated MRSA. Loadings plot from PCA-LDA analysis reveals important functional groups in proteins (1683, 1656, 1596, 1542cm−1), lipids (1743, 1409cm−1), and nucleic acids region of the spectrum (1060, 1087cm−1) that are responsible for the classification of blue light irradiated spectra and control spectra. Cluster vector plots and scores plot reveals that UV light-irradiated spectra are the most biochemically similar to blue light- irradiated spectra; however, some wavenumbers experience a shift. The shifts between blue light and UV light irradiated loadings plot at νasym PO2− band (from 1228 to 1238cm−1), DNA backbone (from 970 to 966cm−1) and base pairing vibration of DNA (from 1717 to 1712cm−1) suggest distinctive changes in DNA conformation in response to irradiation. Our findings indicate that irradiation of MRSA with 470nm light induces A-DNA cleavage and that B-DNA is more resistant to damage by blue light. Blue light and UV light treatment of MRSA are complementary and distinct from the known antimicrobial effect of vancomycin. Moreover, it is known that UV-induced cleavage of DNA predominantly targets B-DNA, which is in agreement with the FTIR findings. Overall the results suggest that the combination of light and vancomycin could be a more robust approach in treating MRSA infections

Investigating the effect of charged amino acids on DNA conformation in EcoRI–DNA complex: a molecular dynamics simulation study

Sequence-specific binding of proteins to DNA is essential for almost all the cellular processes like transcription, translation, replication, etc. One among the various mechanisms that has been identified so far that contributes to the specificity in protein–DNA interaction is the DNA conformational change. Electrostatic neutralization of the phosphate groups by the positively charged amino acid residues in proteins is thought to bring about such conformational changes in DNA. Here, we employ molecular dynamics simulations to examine the effect of charge on amino acids Lys113, Arg145, and Asp91 which are attached to the scissile phosphate on the conformation of DNA in EcoRI–DNA complex. The results indicate that the charge of these amino acids is essential for maintaining the local conformation of DNA in the EcoRI-bound form. Interestingly, we observe that the positively charged amino acids Lys113 and Arg145 have a long-range influence on the DNA conformation, whereas the negatively charged amino acid Asp91 has only a localized effect on the DNA conformation. The charge on the amino acids also alters the collective dynamics of EcoRI. Collectively, the results shed light on the diversity of the effect of charges on DNA conformation as well as on protein dynamics.

Control of the Fluorescence of DNA-templated Silver Nanoclusters by Adenosine Triphosphate and Mercury(II)

Several DNA templates with the sequence 5′-T n TAACCCCTAACCCCT-3′ (n = 0, 15, 30, and 45) were used to prepare DNA template–silver nanoclusters (DNA–Ag NCs). The T n sequence acts as a recognition element for Hg2+, while the rest of the sequence acts as a template for DNA–Ag NCs. At pH 3.0, the fluorescence intensity of DNA–Ag NCs is enhanced by ATP, and the enhanced fluorescence is quenched by Hg2+. The length of polyT shows a slight effect on the sensitivity for the detection of Hg2+ but almost no effect on the optical properties of DNA–Ag NCs. The fluorescence response of DNA–Ag NCs (T15-DNA–Ag NCs) vs. Hg2+ concentration shows two linear ranges over 10–100 and 100–1000 nM, mainly because of the fluorescence quenching due to DNA conformational changes through T–Hg2+–T coordination and the formation of an amalgam with Ag NCs, respectively. The sensitivity of the T15-DNA–Ag NC probe was validated through the analysis of Hg2+ in spiked pond water. Based on the switch-on and switch-off fluorescence properties of T15-DNA–Ag NCs, an IMPLICATION logic gate was fabricated using the concentrations of ATP and Hg2+ as inputs and the fluorescence intensity at 585 nm as output.

Crystal structure and cytotoxic activities of a bis(pyrrolyl-imine) gold(III) complex

A gold(III) complex with N,N′-ethylenebis(pyrrol-2-yl-methyleneamine) (H2pyren) was synthesized and characterized by physicochemical and spectroscopic measurements. Density functional theory (DFT) studies and cytotoxic assays were performed. Infrared, mass spectrometry, and 1H, 13C, and {15N,1H} nuclear magnetic resonance analyses indicate that pyren is deprotonated and gold(III) is four coordinate in a square planar environment, with the pyrrole and imine nitrogens as donors. The structure was confirmed by powder X-ray diffraction and confirmed as a minimum of the potential energy surface by DFT. Cytotoxic activity of [Au(pyren)]+ was active against three tumorigenic cell lines with IC50 values of 35 μM. Interaction studies with CT-DNA by fluorescence and competition with ethidium bromide (EB) showed a quenching of the emission band of DNA with a Stern–Volmer quenching constant value of (3.0 ± 0.1) × 104 M−1 and a decrease in fluorescence quenching of EB-DNA system, respectively, confirming that DNA is a possible target for the complex via an intercalative binding, which was confirmed by DNA conformational changes observed with circular dichroism spectroscopy.

A new dual-signalling electrochemical aptasensor with the integration of “signal on/off” and “labeling/label-free” strategies

The integration of “signal on/off” and “labeling/label-free” strategies was applied to the fabrication of a sensitive and selective dual-signalling electrochemical aptasensor. Ferrocene (Fc), being labeled on single stranded DNA as one signal indicator, could provide “turn on” signal through the DNA conformational changes resulted from target binding with aptamer. [Ru(NH3)6]3+ (RuHex), as another signal indicator, could provide “turn off” signal through the electrostatic interaction between the anionic DNA phosphate backbones and the cationic RuHex, which is a label-free signal. Cyclic voltammetry (CV) and electrochemical impedance spectrum (EIS) were employed for monitoring the stepwise fabrication process of the biosensor. Under the optimized conditions, the dual signal changes were quantified using square wave voltammetry (SWV). Based on the superposition of the dual signal changes (|DIRuHex|+ΔIFc), the sensitivity of this aptasensor for lysozyme detection was improved compared to individual signal. The results indicated that lysozyme could be detected in a wide linear range (1.0×10−11–1.0×10−7M) with a low detection limit down to 0.8 pM. Moreover, the resulting biosensor exhibited good specificity, stability and reproducibility, indicating that the present strategy was promising for broad potential application in clinic assay.