A Single‐Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genomic DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a double‐surface electrochemical technique involving multiple hybridization of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single‐surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen‐printed) electrode. Biotin‐labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidin‐alkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1‐naphthol) which is detected voltammetrically on the same electrode. Signal resulting from this electrochemical enzyme‐linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with osmium tetroxide 2,2′‐bipyridine complex. Detection of (GAA)_n?(TTC)_n triplet repeat expansion in nanogram quantities of PCR‐amplified tDNAs, including amplicons of patients' genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.     

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)₂₅ (DBT) with covalently bound (dT)₂₅ probe are used for the hybridization with target DNA containing adenine stretches. Target DNA is modified with osmium tetroxide,2,2′-bipyridine (Os,bipy) and the immunogenic DNA-Os,bipy adduct is determined by the enzyme-linked immunoassay with electrochemical detection. Electroinactive 1-naphthyl phosphate is used as a substrate and the electroactive product (1-naphthol) is measured on the carbon electrodes. Alternatively Os,bipy-modified target DNA can be determined directly by measuring the osmium signal on the pyrolytic graphite electrode (PGE). A comparison between determinations of the 67-mer oligodeoxynucleotide on carbon electrodes using (a) the guanine oxidation signal, (b) direct determination of the DNA-Os,bipy adduct and (c) its electrochemical immunoassay showed immunoassay to be the most sensitive method. In combination with DBT, the DNA hybridization of long target deoxyoligonucleotides (such as 67- and 97-mers) and a DNA PCR product (226-base pairs) have been detected by immunoassay at high sensitivity and specificity.     

Synthesis and spectroscopic characterization of binaphthol aminosugars for stimulation of DNA strand slippage synthesis

Expansion of DNA repeat sequences is associated with many human genetic diseases. Bulged DNA structures have been implicated as intermediates in DNA slippage within the DNA repeat regions. Two new binaphthol aminosugars were first synthesized as DNA bulge binders to study the triplet repeat expansion due to the wedge-shaped structure of 1,1′-bi-2-naphthol. Both compounds were structurally characterized by 1- and 2-D NMR. They showed remarkable fluorescence enhancement when binding with bulge DNA and they exhibited stimulation for ATT·AAT trinucleotide repeat DNA sequence slippage synthesis.     

Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner

Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.     

Electroanalytical determination of d(GCGAAGC) hairpin

Hairpins (or hairpin-like structures) may play a major role in expansion events of triplet repeat expansion diseases (X syndrome, Huntington's disease, Friedreich's ataxia). The d(GCGAAGC) fragment has been found in the replication origins of phage phiX 174 and herpes simplex virus, in a promoter region of an Escherichia coli heat-shock gene, and in rRNA genes. The paper deals with the application of electrochemical methods to the determination of the DNA heptamer-d(GCGAAGC) which forms very stable hairpin structure in aqueous solutions. On mercury electrodes, this hairpin provides voltammetric reduction signals of adenine and cytosine, and oxidation signals of guanine. Both signals have been studied by cyclic voltammetry (CV), linear sweep voltammetry (LSV), and elimination voltammetry with linear scan (EVLS) in dependence on pH, accumulation time, scan rate, and loop sequences. The EVLS in combination with the adsorptive stripping was employed to the determination of the detection limit (LD) of this mini-hairpin (2 nM). Multidimensional voltammetric data were worked up by Fourier Transform (FT) and for the first coefficient a confidence ellipse was calculated in order to drop out some outlier data. The same method was used also for detection limit determinations. The values of LD obtained by two approaches were compared.     

Accurate and sensitive analysis of triplet repeat expansions by capillary electrophoresis

The reliable genetic diagnostics of triplet repeat expansions by capillary electrophoresis (CE) remains technically challenging due to the properties of the repeated GC-rich sequences. The biased base composition of the analyzed sample as compared to the commonly used DNA size standards makes the precise repeat length determination questionable. The homologous allelic ladders improve the accuracy of the repeat length analysis significantly. Their use, however, is not devoid of other complications. In the approach we propose, the allelic ladders are used only to properly calibrate the commercially available standards which serve then as internal standards in reliable and economical repeat length determination. In this study, we have also analyzed factors that could possibly increase the sensitivity of mutant allele detection by increasing the overall amplification efficiency and long-to-short product ratio.     

Covalent Labeling of Nucleosides with VIII‐ and VI‐Valent Osmium Complexes

Osmium tetroxide complexes with nitrogen ligands [Os(VIII)L] have been widely applied as probes of the DNA structure and as electroactive labels of DNA. Here we describe the electrochemical behavior of Os(VIII)2,2‐bipyridine (Os, bipy)‐base‐labeled nucleosides. We show that electroactive label can be introduced also in the nucleoside ribose residues using six‐valent osmium complex. Cyclic voltammograms of sugar‐Os(VI)‐modified ribosides are similar but not identical to those of the base‐modified ribosides. Our results showing the electroactivity of sugar modified ribosides pave the way to facile end‐labeling of RNA.     

Photophysics of monodisperse platinum-acetylide oligomers: delocalization in the singlet and triplet excited states

A series of monodisperse Pt-acetylide polymers that contain the [-CC-(p-C6H4)-CC-(t-Pt(PBu3)2)-]n repeat unit has been prepared for n = 1, 2, 3, 4, 5, and 7. The photophysical properties of the series provide information concerning the relationship between the oligomer length and delocalization in the singlet and triplet excited states of the pi-conjugated electron system. The results imply that the singlet excited state is delocalized over approximately 6 repeat units; however, the triplet state is considerably more localized. The triplet energy is almost invariant with oligomer length, but the phosphorescence spectra and triplet nonradiative decay rates indicate that the electron-vibrational coupling in the triplet state decreases with increasing oligomer length.     

Electrochemical and electrocatalytic behaviors of poly(ferrocenylsilane)/DNA modified glass carbon electrode

Poly(ferrocenylsilane) (PFS), composed of electroactive ferrocene units existing in the polymer main chain, was directly dropped on the DNA modified glassy carbon electrode (GCE). The electrochemical and electrocatalytic behaviors of PFS/DNA/GCE were investigated in detail. Cyclic voltammograms of the PFS/DNA/GCE showed two pairs of reversible redox peaks, indicating a stepwise oxidation of the electroactive units. It was found that DNA film played a key role in immobilizing the PFS and facilitating the electron transfer between electroactive units of PFS and the GCE surface. Moreover, the PFS/DNA/GCE showed a good electrocatalytic ability to the oxidization of ascorbic acid (AA), suggesting it had the potential application as a new type of sensor to detect AA.     

Sensing mispaired thymines in DNA heteroduplexes using an electroactive osmium marker: towards electrochemical SNP probing

A complex OsO₄, 2,2′-bipyridine (Os,bipy), has been used for electroactive labeling of biopolymers as well as for probing of nucleic acids and protein structure and interactions. In DNA, Os,bipy forms electrochemically active adducts with pyrimidine nucleobases, exhibiting highly selective modification of thymine residues in single-stranded DNA. Here, we show that modification of rare thymine residues (one thymine among several tens of unreactive purine bases) can easily be detected by means of a simple ex situ voltammetric analysis using carbon electrodes. Based on this remarkable sensitivity of detection, Os,bipy has been used as an electroactive probe for unpaired and/or mismatched thymine residues within DNA heteroduplexes. Site-specific chemical modification of the DNA with the Os,bipy has allowed a clear distinction between perfectly base-paired DNA homoduplexes and mismatched heteroduplexes, as well as discrimination among heteroduplexes containing one or two mispaired thymines, a single thymine insertion, or combination of a mispair and an insertion.     

Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode

In this paper, we present an electrochemical DNA–protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT₂₀ tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed. Figure

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New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces

Up to now, the development of the electrochemical DNA hybridization sensors relied on solid electrodes, on which both the hybridization and detection steps have been performed. Here we propose a new method in which the DNA hybridization is performed at commercially available magnetic beads and electrochemical detection on detection electrodes (DE). Due to minimum nonspecific DNA adsorption at the magnetic beads, very high specificity of the DNA hybridization is achieved. Optimum DE can be chosen only with respect to the given electrode process. It is shown that high sensitivity and specificity in the detection of relatively long target DNAs can be obtained (a) by using cathodic stripping voltammetry at mercury or solid mercury amalgam DEs for the determination of purine bases, released from DNA by acid treatment, and (b) by enzyme-linked immunoassay of target DNA modified by osmium tetroxide,2,2'-bipyridine (Os,bipy) at carbon DEs. Direct determination of Os,bipy at mercury and carbon electrodes is also possible.     

Voltammetric Behavior of Osmium‐Labeled DNA at Mercury Meniscus‐Modified Solid Amalgam Electrodes. Detecting DNA Hybridization

The complex of osmium tetroxide with 2,2′‐bipyridine has been utilized as a probe of DNA structure and an electroactive marker of DNA in DNA hybridization sensors. It produces several voltammetric signals, the most negative of them has been observed only at mercury electrodes. This signal is of catalytic nature affording a high sensitivity of DNA determination. The catalytic current due to evolution of hydrogen in voltammetry of DNA modified by complex of osmium tetroxide with 2,2′‐bipyridine (DNA‐Os,bipy) was studied. Solid amalgam electrodes (modified with mercury menisci) of silver (m‐AgSAE), copper (m‐CuSAE), gold, and of combined bismuth and silver, were used as possible substitutes for mercury electrodes. Besides the hanging mercury drop electrode (HMDE), the catalytic current was observed only on m‐AgSAE and m‐CuSAE. Electrodes of gold and bismuth amalgams did not give the catalytic current. The detection limit of DNA‐Os,bipy on HMDE was 0.1 ng mL^?1 (RSD=2.3 %, N=11), and on m‐AgSAE 0.2 ng mL^?1 (RSD=3.1%, N=11). The m‐AgSAE was successfully applied as a detection electrode in double‐surface DNA hybridization experiments offering highly specific discrimination between complementary (target) and nonspecific DNAs, as well as determination of the length of a repetitive DNA sequence. The m‐AgSAE has proved a convenient alternative to the HMDE or carbon electrodes used for similar purposes in previous work.     

Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as "rollamers". Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain ("rollamerization"). We demonstrate that such "rollameric" migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor "rollamers" with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic "universal hinge" relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.     

The influence of sequence context and length on the kinetics of DNA duplex formation from complementary hairpins possessing (CNG) repeats

The formation of unusual structures during DNA replication has been invoked for gene expansion in genomes possessing triplet repeat sequences, CNG, where N = A, C, G, or T. In particular, it has been suggested that the daughter strand of the leading strand partially dissociates from the parent strand and forms a hairpin. The equilibrium between the fully duplexed parent:daugter species and the parent:hairpin species is dependent upon their relative stabilities and the rates of reannealing of the daughter strand back to the parent. These stabilities and rates are ultimately influenced by the sequence context of the DNA and its length. Previous work has demonstrated that longer strands are more stable than shorter strands and that the identity of N also influences the thermal stability [Paiva, A. M.; Sheardy, R. D. Biochemistry 2004, 43, 14218-14227]. Here, we show that the rate of duplex formation from complementary hairpins is also sequence context and length dependent. In particular, longer duplexes have higher activation energies than shorter duplexes of the same sequence context. Further, [(CCG):(GGC)] duplexes have lower activation energies than corresponding [(CAG):(GTC)] duplexes of the same length. Hence, hairpins formed from long CNG sequences are more thermodynamically stable and have slower kinetics for reannealing to their complement than shorter analogues. Gene expansion can now be explained in terms of thermodynamics and kinetics.     

DNA microspot assay using single-molecule detection and requiring 1.8 nL samples only

An ultra-sensitive DNA microspot assay was developed that required 1.8?nL samples and was based on single-molecule detection. The solution of the target DNA (tDNA) was spotted onto the coverslip modified with capture DNA (DNA1) and blocked with ethanolamine and bovine serum albumin using a pintool type microspoting robot. The microspot had a diameter of ~300???m. The tDNA was captured by the DNA1, and the tDNA was then labeled with a detection DNA that previously was labeled with a quantum dot. Next, a fluorescence microscopic image of the microspot was acquired using a single-molecule microspot reader during total internal reflection fluorescence excitation. As little as 4?×?10^?22 mole (240 molecules) of tDNA can be detected by this method. The response is linear in the range from 6.0?×?10^?22 to 1.2?×?10^?19 mole of tDNA. All operations (including the acquisition of microspot images and single-molecule counting) were performed using the MetaMorph software. The assay was applied to the determination of osteopontin messenger RNA in single decidual stromal cells without the need for PCR amplification.

Electrochemical detection of short sequences related to the hepatitis B virus using MB on chitosan-modified CPE

A novel electrochemical DNA biosensor based on methylene blue (MB) and chitosan-modified carbon paste electrode (CCPE) for short DNA sequences and polymerase chain reaction (PCR) amplified real samples related to the hepatitis B virus (HBV) hybridization detection is presented. Differential pulse voltammetry (DPV) was used to investigate the surface coverage and hybridization event. The decrease in the peak current of MB, an electroactive label, was observed upon hybridization of probe with the target. Numerous factors affecting the target hybridization and indicator binding reaction are optimized to maximize the sensitivity.     

An ultra-sensitive DNA assay based on single-molecule detection coupled with hybridization accumulation and its application

An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10(-18) mol L(-1). Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.     

On the conjugation length for oligo(ethynylnaphthalene)-based molecular rods

The synthesis is described for a small series of oligomers built from (2, 3, 4 or 6) ethynyl-naphthalene repeat units and end-capped with solubilising 1,2,3-tris-dodecyloxy-benzene groups. These compounds absorb in the near-UV region and exhibit strong fluorescence in both fluid solution and a glassy matrix at 77 K. The spectral profiles are fully consistent with a structurally heterogeneous ground state becoming more planar upon excitation and with the low-temperature glass further stabilising the co-planar orientation. The absorption and fluorescence maxima move towards lower energy with increasing number of repeat units and there is a corresponding increase in the Huang-Rhys factor for the radiative process. The non-radiative rate constants also depend on molecular length and are well explained in terms of the energy-gap law. In contrast, very weak phosphorescence is observed at 77 K for which the peak maximum and lifetime remain insensitive to the number of naphthalene units. The triplet lifetimes recorded at ambient temperature are also independent of the molecular length but the triplet-triplet absorption spectra change throughout the series. These results are discussed in terms of the degree of electronic coupling between adjacent repeat units for each of the relevant excited states. During these studies it was noted that the rate of intersystem crossing to the triplet manifold is but weakly affected by heavy-atom perturbers. A non-fluorescent complex is formed between iodoethane and the molecular rod but the corresponding bimolecular process occurs at well below the diffusion-controlled limit. This behaviour is considered in terms of spin-orbit coupling between the excited states and takes account of the differing conjugation lengths.     

血清样品中乙肝病毒的DNA电化学传感器检测

利用自组装单分子膜技术，将巯己基修饰的具有乙肝病毒(HBV)DNA序列特异性的单链DNA探针固定在金电极表面，制得DNA电化学传感器；以电活性的Hoechst 33258为指示剂，考察了该传感器对血清样品中乙肝病毒DNA的响应；探讨DNA电化学传感器在临床检测中的应用；将传感器法与荧光聚合酶链反应(PCR)法进行对比，两者的分析结果具有一致性。