Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector.

Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone. Lesions were positioned on opposite strands of the vector. When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion. Mutagenesis occurred in the absence of SOS induction. Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure. For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed. dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector. We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells. Repair DNA synthesis may be involved in this process.     

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.     

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.     

NMR characterization of clustered bistrand abasic site lesions: effect of orientation on their solution structure

A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction of clustered damage: two or more DNA lesions (oxidized bases, abasic sites, or strand breaks) occurring in the same or different strands of the DNA molecule within a single turn of the helix. In spite of arising at a lower frequency than single lesions, clustered DNA damage represents an exotic challenge to the repair systems present in the cells and, in some cases, these lesions may escape detection and/or processing. To understand the structural properties of clustered DNA lesions we have prepared two oligodeoxynucleotide duplexes containing adjacent tetrahydrofuran residues (abasic site analogues), positioned one in each strand of the duplex in a 5' or 3' orientation, and determined their solution structure by NMR spectroscopy and molecular dynamics simulations. The NMR data indicate that both duplex structures are right-handed helices of high similarity outside the clustered damage site. The thermal stability of the duplexes is severely reduced by the presence of the abasic residues, especially in a 5' orientation where the melting temperature is 5 degrees C lower. The structures show remarkable differences at the lesion site where the extrahelical location of the tetrahydrofuran residues in the (AP)(2)-5'-staggered duplex contrasts with their smooth alignment along the sugar-phosphate backbone in the (AP)(2)-3'-staggered duplex.     

Condensation of DNA—A putative obstruction for repair process in abasic clustered DNA damage

Clustered DNA damages are defined as two or more closely located DNA damage lesions that may be present within a few helical turns of the DNA double strand. These damages are potential signatures of ionizing radiation and are often found to be repair resistant. Types of damaged lesions frequently found inside clustered DNA damage sites include oxidized bases, abasic sites, nucleotide dimers, strand breaks or their complex combinations. In this study, we used a bistranded two-lesion abasic cluster DNA damage model to access the repair process of DNA in condensate form.Oligomer DNA duplexes (47 bp) were designed to have two deoxyuridine in the middle of the sequences, three bases apart in opposite strands. The deoxyuridine residues were converted into abasic sites by treatment with UDG enzyme creating an abasic clustered damage site in a precise position in each of the single strand of the DNA duplex. This oligomer duplex having compatible cohesive ends was ligated to pUC19 plasmid, linearized with HindIII restriction endonuclease. The plasmid–oligomer conjugate was transformed into condensates by treating them with spermidine. The efficiency of strand cleavage action of ApeI enzyme on the abasic sites was determined by denaturing PAGE after timed incubation of the oligomer duplex and the oligomer–plasmid conjugate in presence and absence of spermidine. The efficiency of double strand breaks was determined similarly by native PAGE. Quantitative gel analysis revealed that rate of abasic site cleavage is reduced in the DNA condensates as compared to the oligomer DNA duplex or the linear ligated oligomer–plasmid conjugates. Generation of double strand break is significantly reduced also, suggesting that their creation is not proportionate to the number of abasic sites cleaved in the condensate model. All these suggest that the ApeI enzyme have difficulty to access the abasic sites located deep into the condensates leading to repair refractivity of the damages. In addition, we found that presence of a polyamine such as spermidine has no notable effect in the incision activity of ApeI enzyme in linear oligomer DNA duplexes in our experimental concentration.     

A simple, non-nucleosidic base surrogate increases the duplex stability of DNA containing an abasic site

Abasic sites represent a common type of lesion in DNA. If not repaired, they can lead to mutations during replication or to cell death. Due to their biological importance, there is a strong interest in methods of recognizing abasic sites in DNA both for diagnostic and also for potential pharmaceutical applications. Extended aromatic residues can have a substantial positive influence on the stability of double-stranded DNA containing abasic sites. We report here the use of a simple, non-nucleosidic phenanthrene as a base surrogate, which effectively enhances the duplex stability of DNA with an abasic site. The influence of the linker length on the stability of the duplex is investigated. Data and model considerations indicate that stabilization is a result of stacking interactions between the phenanthrene and DNA base pairs.     

NMR solution structures of bistranded abasic site lesions in DNA

Ionizing radiation produces clustered lesions in DNA. Since the orientation of bistranded lesions affects their recognition by DNA repair enzymes, clustered damages are more difficult to process and thus more toxic than single oxidative lesions. In order to understand the structural determinants that lead to differential recognition, we used NMR spectroscopy and restrained molecular dynamics to solve the structure of two DNA duplexes, each containing two stable abasic site analogues positioned on opposite strands of the duplex and staggered in the 3' (-1 duplex, (AP) 2-1 duplex) or 5' (+1 duplex, (AP) 2+1 duplex) direction. Cross-peak connectivities observed in the nonexchangeable NOESY spectra indicate compression of the helix at the lesion site of the duplexes, resulting in the formation of two abasic bulges. The exchangeable proton spectra show the AP site partner nucleotides forming interstrand hydrogen bonds that are characteristic of a Watson-Crick G.C base pairs, confirming the extra helical nature of the AP residues. Restrained molecular dynamics simulations generate a set of converging structures in full agreement with the spectroscopic data. In the (AP) 2-1 duplex, the extra helical abasic site residues reside in the minor groove of the helix, while they appear in the major groove in the (AP) 2+1 duplex. These structural differences are consistent with the differential recognition of bistranded abasic site lesions by human AP endonuclease.     

Effects of the presence of an aldehydic abasic site on the thermal stability and rates of helix opening and closing of duplex DNA.

The presence of an abasic site in duplex DNA lowers the thermodynamic stability, as monitored by the optical melting temperature, and decreases the rate of imino proton exchange with water, by about an order of magnitude, as monitored by direct measurement of both the exchange lifetimes and the imino proton T1S. The exchange lifetimes of the imino protons with water as a function of base catalyst concentration were analyzed to determine the origin of the effect of the abasic site on imino exchange lifetimes. Analysis of the results showed that the helix opening rate is not significantly changed by the presence of an abasic site. The differences in exchange lifetimes are attributed to a faster helix closing rate in the presence of an abasic site. The faster rate of helix closing may be an important contribution to the stability of abasic sites in duplex DNA to base-catalyzed elimination reaction. It is noted that duplex DNAs containing analogues of the aldehydic abasic site apparently do not exhibit these exchange lifetime effects.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N2-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9).(G10-T11-A12-C-13-F14-C15 -A16-T17-G-18) X.F 9-mer duplex. Two-dimensional NMR experiments establish that the X.F 9-mer helix is right-handed with Watson-Crick A.T and G.C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4.C15 and G6.C13 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4.C15 and G6.C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N2-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.     

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N ²-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.     

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.     

Characterization of conformational features of DNA heteroduplexes containing aldehydic abasic sites

The DNA duplexes shown below, with D indicating deoxyribose aldehyde absic sites and numbering from 5' to 3', have been investigated by NMR. The 31P and 31P-1H correlation data indicate [formula: see text] that the backbones of these duplex DNAs are regular. One- and two-dimensional 1H NMR data indicate that the duplexes are right-handed and B-form. Conformational changes due to the presence of the abasic site extend to the two base pairs adjacent to the lesion site with the local conformation of the DNA being dependent on whether the abasic site is in the alpha or beta configuration. The aromatic base of residue A17 in the position opposite the abasic site is predominantly stacked in the helix as is G17 in the analogous sample. Imino lifetimes of the AT base pairs are much longer in samples with an abasic site than in those containing a Watson-Crick base pair. The conformational and dynamical properties of the duplex DNAs containing the naturally occurring aldehyde abasic site are different from those of duplex DNAs containing a variety of analogues of the abasic site.     

Product inhibition and magnesium modulate the dual reaction mode of hOgg1

8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3' to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.     

Temperature and Electrolyte Optimization of the α-Hemolysin Latch Sensing Zone for Detection of Base Modification in Double-Stranded DNA

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol⁻¹ decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.     

Nucleosides and oligonucleotides containing 1,2,3-triazole residues with nucleobase tethers: synthesis via the azide-alkyne 'click' reaction

A series of novel 1,2,3-triazole nucleosides linked to DNA nucleobases were prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurines or N-1 propargylpyrimidines with the tolouyl protected 1-azido-2-deoxyribofuranose 2 followed by treatment with NaOMe/MeOH or aq NH3. The antiviral activity of such compounds against selected RNA viruses is reported. The strongly fluorescent 1,2,3-triazole compounds 16 and 17 were synthesized from propargylated uracil 1a and propargylated adenine 1c with coumarin azide 15, and the fluorescence properties were studied. The nucleosides 4 and 6 were incorporated into DNA using the phosphoramidite building blocks and employed in solid-phase synthesis. Melting experiments demonstrated that such 1,2,3-triazole nucleosides have a negative impact on the duplex stability when they are placed opposite to the canonical bases as well as abasic sites. The nucleobases attached to the triazole ring cannot involve in the base pair formation with the opposite bases because of the structural variations induced by the triazole ring. The stacking of such nucleosides when positioned at the end of oligonucleotides retains the stability of DNA duplexes. The duplex structures were studied by molecular modelling which support the results of melting experiments.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P-DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 microM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10(6) bp versus 3.7 lesions/10(6) bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3-10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mbeta16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P-DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Impact of the C1' configuration of abasic sites on DNA duplex structure

Naturally occurring abasic sites in DNA exist as an equilibrium mixture of the aldehyde, the hydrated aldehyde, and the hemiacetal forms (dominant). The influence of the configuration of the C1' hydroxyl group of the hemiacetal form on duplex structure and abasic site repair has been examined using novel carbocyclic analogues. Both the alpha- and beta-forms of this novel abasic site were introduced into oligomeric DNA using the standard DMT-phosphoramidite approach in an automated solid-phase synthesizer. Solution structures of the d(CGTACXCATGC).d(GCATGAGTACG) duplex (where X is the alpha- or beta-anomer of the carbocyclic abasic site analogue) were determined by NMR spectroscopy and restrained molecular dynamics simulations. The structures were only minimally perturbed by the presence of either anomer of the abasic site. All residues adopted an anti conformation, and Watson-Crick alignments were observed on all base pairs of the duplexes. At the lesion site, the abasic residues and their partner adenines showed increased dynamic behavior but adopted intrahelical positions in the final refined structures. Incision of duplexes having the alpha- or beta-anomer of the carbocyclic abasic site by human AP endonuclease showed that the enzyme recognizes both configurations of the lesion and nicks the DNA backbone with similar efficiency. Our results challenge the suggestion that Ape1 is stereoselective and imply a plasticity at the active site of the enzyme for accommodating either anomer of the lesion.     

The impact of abasic sites on DNA flexibility

We use internal coordinate molecular mechanics calculations to study the impact of abasic sites on the conformation and the mechanics of the DNA double helix. Abasic sites, which are common mutagenic lesions, are shown to locally modify both the groove geometry and the curvature of DNA in a sequence dependent manner. By controlled twisting and bending, it is also shown that these lesions modify the deformability of the duplex, generally increasing its flexibility, but again to an extent which depends on the nature of the abasic site and on the surrounding base sequence. Both the conformational and mechanical influence of this type of DNA damage may be significant for recognition and repair mechanisms.     

CuO thin film based uric acid biosensor with enhanced response characteristics

An electrochemical approach for detection of individual single nucleotide polymorphisms (SNPs) based on nucleobase-conjugated apoferritin probe loaded with metal phosphate nanoparticles is reported. Coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA was induced by DNA polymerase I (Klenow fragment) to preserve Watson-Crick base-pairing rules. After sequential liquid hybridization of biotinylated DNA probes with mutant DNA and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a well known and specific biotin-streptavidin affinity binding. For signaling each of eight possible Single-nucleotide polymorphisms (SNPs), Pb, Cu, Cd and Zn phosphate-loaded apoferritin nanoparticle probes were linked to adenosine (A), cytidine (C), guanosine (G), and thymidine (T) mononucleotides, respectively. Monobase-conjugated apoferritin probes were coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Electrochemical stripping analyses of the metals loaded in apoferritin nanoparticle probes provide a means for detection and quantification of mutant DNA. Each mutation captures different nucleotide-conjugated apoferritin probe and provide a distinct four-potential voltammogram, whose peak potentials reflect the identity of the mismatch. The method is sensitive enough to accurately determine AG mutation, as the most thermodynamically stable mismatch to detect, in the range of 50-600 pM. The proposed protocol provides a simple, fast, cost-effective, accurate and sensitive method for detection of SNPs.