Klenow Fragment Exo Research Articles

Analysis of nucleotide insertion opposite urea and translesion synthesis across urea by DNA polymerases

Urea (Ua) is produced in DNA as the result of oxidative damage to thymine and guanine. It was previously reported that Klenow fragment (Kf) exo− incorporated dATP opposite Ua, and that DNA polymerase β was blocked by Ua. We report here the following nucleotide incorporations opposite Ua by various DNA polymerases: DNA polymerase α, dATP and dGTP (dATP > dGTP); DNA polymerase δ, dATP; DNA polymerase ζ, dATP; Kf exo−, dATP; Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), dGTP and dATP (dGTP > dATP); and DNA polymerase η, dCTP, dGTP, dATP, and dTTP (dCTP > dGTP > dATP > dTTP). DNA polymerases β and ε were blocked by Ua. Elongation by DNA polymerases δ and ζ stopped after inserting dATP opposite Ua. Importantly, the elongation efficiency to full-length beyond Ua using DNA polymerase η and Dpo4 were almost the same as that of natural DNA.Graphical abstract

Double‐Stranded DNA‐Templated Copper Nanoclusters for Detection of DNA Polymerase Activity

DNA polymerase plays an important role in the proliferation and survival of all forms of life and the variety of biotechnological applications. In this study, DNA‐templated copper nanoclusters were utilized to detect Klenow fragment exo‐ (KF‐) activity and monitor its inhibition in the presence of 5‐fluorouracil by measurement of fluorescence intensities. This study showed that the fluorescence intensities increased continuously for KF‐ concentrations ranging from 0 to 5 U/mL. In addition, inhibition of KF‐ activity was measured at 5‐fluorouracil concentrations ranging from 0 to 150 μM. Using nonlinear regression, the corresponding IC50 was determined to be 6.23 μM in the presence of 5.0 U/mL KF‐.

Ultrasensitive microchip electrophoretic detection of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) based on isothermal strand-displacement polymerase reaction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens involved in hospital and community infection. To rapidly and sensitively detect the mecA gene, which is relevant to methicillin-resistant strains, microchip electrophoresis (MCE) integrated with isothermal strand-displacement polymerase reaction (ISDPR) was developed. In the ISDPR signal recycle amplification, the target DNA opened the DNA hairpin structure by specifically binding with the hairpin probe (HP), and then the primer hybridized with the probe and released the target DNA in the presence of Klenow Fragment exo− (KF exo−) polymerase. The released target DNA hybridized with the next HP and then was displaced by the primer again, consequently achieving target recycling and amplification. The amplified products of the HP-cDNA duplex were separated rapidly from other DNAs by MCE. Under optimal conditions, the limit of detection of the target DNA was as low as 12.3 pM (S/N = 3). The proposed ISDPR with MCE method was also successfully applied to detect methicillin-resistant S. aureus, and the experimental results showed that it had some advantages such as being label free, ultrasensitive, rapid and well separated.

Synthesis of Deoxypseudouridine 5'-Triphosphate Bearing the Photoremovable Protecting Group at the N1 Position Capable of Enzymatic Incorporation to DNA.

Enzymatic incorporation of deoxynucleoside 5'-triphosphate bearing the photocleavable protecting group is a useful method for the preparation of photocaged oligodeoxynucleotides. Here, we describe the synthesis of new photocaged deoxynucleoside triphosphates N1-(2-nitrobenzyl)-deoxypseudouridine triphosphate (dNBΨTP) and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine triphosphate (dNPOMΨTP). We successfully synthesized dNBΨTP and dNPOMΨTP and applied them to enzymatic synthesis of photocaged oligonucleotides. In addition, we also synthesized phosphoramidites of N1-(2-nitrobenzyl)- and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine to enable chemical synthesis of photocaged oligonucleotides incorporating them. The photocleavable 2-nitrobenzyl and 6-nitropiperonyloxymethyl in oligonucleotides were cleaved by irradiation at 365 nm for 30 and 10 s, respectively. We also studied the enzymatic incorporation of dNBΨTP and dNPOMΨTP using the Klenow fragment exo-. As a result, it was clarified that dNPOMΨTP could be incorporated to oligonucleotide 193 times more efficiently than dNBΨTP, as judged by Vmax/Km. We also performed the incorporation of at least eight dNPOMΨ residues in a 35-mer oligodeoxynucleotide. It has also been revealed that the oligodeoxynucleotides incorporating photocaged deoxypseudouridine were useful for photocontrol of DNA triplex formation.

Label-Free Detection of DNA Polymerase Activity Using Hairpin DNA-Templated Silver Nanoclusters.

Based on the characteristic properties like strong fluorescence and high photostability, DNA-templated silver nanoclusters (DNA-AgNCs) were utilized to detect Klenow fragment exo- (KF-) activity and to monitor its inhibition by daidzein. In this study, fluorescence intensity is linearly correlated for KF- concentration ranging from 0 to 2.0 U/mL, with a minimum detectable concentration of 0.05 U/mL. In addition, KF- inhibition was monitored by changes in fluorescence intensity with increasing daidzein concentration gradually.

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2'-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5'-GGCXCC-3', where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (-2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats ( NarI: 3'-CXCGGC-5' and CG3: 3'-CXCGCG-5', X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo- (Kf-) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf- extended the primer past FBP-dG within a CG3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on -2 frameshift mutations mediated by the C-linked FBP-dG lesion.

Deoxynucleoside Triphosphate Containing Pyridazin-3-one Aglycon as a Thymidine Triphosphate Substitute for Primer Extension and Chain Elongation by Klenow Fragments.

Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2 H-pyridazin-6-yl (PzO)-a uracil analogue lacking a 2-keto group-as the nucleobase. Theoretical analyses and hybridization experiments indicated that PzO recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside 5'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside 5'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3'-nucleotide residue harboring PzO as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the PzO base at the 3' end could function in subsequent chain elongation. These properties of PzO were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.

Luminescence determination of microRNAs based on the use of terbium(III) sensitized with an enzyme-activated guanine-rich nucleotide.

A method is reported for the fluorometric quantitation of microRNA. It is making use of a luminescent probe deribed from terbium(III) ion whose fluorescence is sensitized with a guanine-rich (G-rich) nucleotide. The probe has a large Stokes' shift and strong and sharp emission bands. The assay relies on the wide substrate specificity of terminal deoxynucleotidyl transferase (TdTase), which catalyzes the formation of long G-rich nucleotides when using microRNA primer as a trigger to start the polymerization. The addition of Tb(III) induces the formation of a G-quadruplex from the G-rich nucleotide, and this strongly enhances the green fluorescence of Tb(III) (peaking at 545nm upon photoexcitation at 290nm). Specifically, microRNA-21 was chosen as the analyte. The fluorescence intensity of Tb(III) increases linearly in the 1 pM to 1nM microRNA concentration range, and the detection limit is as low as 0.11 pM. The method can distinguish between family members of microRNA and performs excellently even when applied to extracts of cancer cells. Graphical abstract A fluorometric technique is reported for the determination of microRNA. It is based on signal enhancement based on the sensitization of terbium(III) via a guanine-rich nucleotide sequence. Klenow Fragment exo- (KFexo-) generates DNA sequence at the 3'-OH of microRNA, and terminal deoxynucleotidyl transferase (TdTase) catalyzes the formation of long G-rich nucleotides.

Programmable strand displacement-based magnetic separation for simultaneous amplified detection of multiplex microRNAs by chemiluminescence imaging array

High throughput analysis of miRNAs is of great significance to clinical and biomedical applications. In this work, we have developed a highly sensitive and selective chemiluminescence imaging array (CLIA) for simultaneous detection of three miRNAs with high throughput, easy operation and low cost. In this assay, three kinds of hairpins that can specially recognize respective miRNAs (miR-155, miR-let-7a and miR-141) are parallel modified on the magnetic beads (MBs). The employment of MBs enables quick sorting of multiplex targets in high yield and purity with high throughput. Upon introduction of the target miRNAs, they hybridize to the corresponding hairpins and initiate the DNA machines with the assistance of Klenow fragment exo- DNA polymerase and Nb.BbvCI NEase, achieving exponential amplification of targets. After that, the toehold-mediated strand displacement (TMSD) reactions are performed through sequential addition of displacement probes under magnetic separation, resulting in the release of HRP-tagged DNA hybrids in solution for CL imaging and recovery of MBs for repetitive use. This CLIA method demonstrates ultrahigh sensitivity with detection limits down to fM level, wide linear range over 5 orders of magnitude, excellent selectivity to distinguish one-base mismatched target miRNA, and good performance in real sample analysis. Together with the advantages of high throughput, easy operation, acceptable accuracy, and good recyclability and reproducibility, the proposed multiplex CLIA method holds great potential in practical applications, such as profiling the pattern of miRNA expression, disease screening, biomedical research, and so on.

Time Course Analysis of Enzyme-Catalyzed DNA Polymerization.

Extracting kinetic parameters from DNA polymerase-catalyzed processive polymerization data using traditional initial-rate analysis has proven to be problematic for multiple reasons. The first substrate, DNA template, is a heterogeneous polymer and binds tightly to DNA polymerase. Further, the affinity and speed of incorporation of the second substrate, deoxynucleoside triphosphate (dNTP), vary greatly depending on the nature of the templating base and surrounding sequence. Here, we present a mathematical model consisting of the DNA template-binding step and a Michaelis-Menten-type nucleotide incorporation step acting on a DNA template with a finite length. The model was numerically integrated and globally fitted to experimental reaction time courses. The time courses were determined by monitoring the processive synthesis of oligonucleotides of lengths between 50 and 120 nucleotides by DNA polymerase I (Klenow fragment exo-) using the fluorophore PicoGreen. For processive polymerization, we were able to estimate an enzyme-template association rate k1 of 7.4 μM-1 s-1, a disassociation rate k-1 of 0.07 s-1, and a Kd of 10 nM, and the steady-state parameters for correct dNTP incorporation give kcat values of 2.5-3.3 s-1 and Km values of 0.51-0.86 μM. From the analysis of time courses measured between 5 and 25 °C, an activation energy for kcat of 82 kJ mol-1 was calculated, and it was found that up to 73% of Klenow fragment becomes inactivated or involved in unproductive binding at lower temperatures. Finally, a solvent deuterium kinetic isotope effect (KIE) of 3.0-3.2 was observed under processive synthesis conditions, which suggests that either the intrinsic KIE is unusually high, at least 30-40, or previous findings, showing that the phosphoryl transfer step occurs rapidly and is flanked by two slow conformational changes, need to be re-evaluated. We suggest that the numerical integration of rate equations provides a high level of flexibility and generally produces superior results compared to those of initial-rate analysis in the study of DNA polymerase kinetics and, by extension, other complex enzyme systems.

Direct fluorescence detection of microRNA based on enzymatically engineered primer extension poly-thymine (EPEPT) reaction using copper nanoparticles as nano-dye

A new strategy based on enzymatically engineered primer extension poly-thymine (EPEPT) and nanomaterials in situ generation technology is reported for direct detection of microRNA (miRNA) in a fluorescence turn-on format using the sequential and complementary reactions catalyzed by Klenow Fragment exo− (KFexo−) and terminal deoxynucleotidyl transferase (TdTase). The short miRNA can be efficiently converted into long poly-thymine (polyT) sequences, which function as template for in situ formation of fluorescence copper nanoparticles (CuNPs) as nano-dye for detecting miRNA. The polyT-CuNPs can effectively form and emit intense red fluorescence under the 340nm excitation. For the proof of concept, microRNA-21 (miR-21) was selected as the model target to testify this strategy as a versatile assay platform. By directly using miR-21 as the primer, the simple, rapid and sensitive miRNA detection was successfully achieved with a good linearity between 1pM and 1nM and a detection limit of 100fM. Thus, the EPEPT strategy holds great potential in biochemical sensing research as an efficient and universal platform.

Label-free simple fluorescence assay of DNA polymerase using the G-quadruplex structure

In this study, the G-quadruplex structure was used to detect the enzymatic activity and inhibition of the Klenow fragment exo−(KF−) in the presence ofN-methylmesoporphyrin IX (NMM) which binds specifically to the G-quadruplex.

PH-Dependent Equilibrium between 5-Guanidinohydantoin and Iminoallantoin Affects Nucleotide Insertion Opposite the DNA Lesion.

Four-electron oxidation of 2'-deoxyguanosine (dG) yields 5-guanidinohydantoin (dGh) as a product. Previously, we hypothesized that dGh could isomerize to iminoallantoin (dIa) via a mechanism similar to the isomerization of allantoin. The isomerization reaction was monitored by HPLC and found to be pH dependent with a transition pH = 10.1 in which dGh was favored at low pH and dIa was favored at high pH. The structures for these isomers were confirmed by UV-vis, MS, and (1)H and (13)C NMR. Additionally, the UV-vis and NMR experimental results are supported by density functional theory calculations. A mechanism is proposed to support the pH dependency of the isomerization reaction. Next, we noted the hydantoin ring of dGh mimics thymine, while the iminohydantoin ring of dIa mimics cytosine; consequently, a dGh/dIa site was synthesized in a DNA template strand, and standing start primer extension studies were conducted with Klenow fragment exo(-). The dATP/dGTP insertion ratio opposite the dGh/dIa site as a function of pH was evaluated from pH 6.5-9.0. At pH 6.5, only dATP was inserted, but as the pH increased to 9.0, the amount of dGTP insertion steadily increased. This observation supports dGh to dIa isomerization in DNA with a transition pH of ∼8.2.

Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo− (KF exo−) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo− and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3′ Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3′ or 5′ Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.

Guanine oxidation product 5-carboxamido-5-formamido-2-iminohydantoin induces mutations when bypassed by DNA polymerases and is a substrate for base excision repair.

Guanine (G) is a target for oxidation by reactive oxygen species in DNA, RNA, and the nucleotide pool. Damage to DNA yields products with alternative properties toward DNA processing enzymes compared to those of the parent nucleotide. A new lesion, 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), bearing a stereocenter in the base was recently identified from the oxidation of G. DNA polymerase and base excision repair processing of this new lesion has now been evaluated. Single nucleotide insertion opposite (S)-2Ih and (R)-2Ih in the template strand catalyzed by the DNA polymerases Klenow fragment exo(-), DPO4, and Hemo KlenTaq demonstrates these lesions to cause point mutations. Specifically, they promote 3-fold more G·C → C·G transversion mutations than G·C → T·A, and (S)-2Ih was 2-fold more blocking for polymerase bypass than (R)-2Ih. Both diastereomer lesions were found to be substrates for the DNA glycosylases NEIL1 and Fpg, and poorly excised by endonuclease III (Nth). The activity was independent of the base pair partner. Thermal melting, CD spectroscopy, and density functional theory geometric optimization calculations were conducted to provide insight into these polymerase and DNA glycosylase studies. These results identify that formation of the 2Ih lesions in a cell would be mutagenic in the event that they were not properly repaired.

Synthesis of C8-N-Arylamine-Modified 2'-Deoxyguanosine-5'-Triphosphates and Their Effects on Primer Extension by DNA Polymerases.

C8-N-arylamine adducts of 2'-deoxyguanosine (2'-dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8-N-acetyl-N-arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8-N-acetyl-N-arylamine-2'-deoxyguanosine-5'-triphosphates and C8-NH-arylamine-2'-deoxyguanosine-5'-triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8-dG adducts were studied in primer-extension assays by using Klenow fragment exo(-) of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N-acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8-N-arylamine-2'-deoxyguanosine-5'-triphosphates in chemical carcinogenesis.

Chlorine Functionalization of a Model Phenolic C8-Guanine Adduct Increases Conformational Rigidity and Blocks Extension by a Y-Family DNA Polymerase

Certain phenoxyl radicals can attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure-activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on in vitro DNA replication by Klenow fragment exo(-) (Kf(-)) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]G) and 2,4,6-trichlorophenoxyl radical ([TCP]G) were inserted into the reiterated G3-position of the NarI sequence (12-mer, NarI(12); and 22-mer, NarI(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the NarI(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]G to afford [PCP]G does not significantly change the adduct conformation within the NarI(12) duplex, as predicted by molecular dynamics simulations. However, when using NarI(22) for DNA synthesis in vitro, the chlorinated [PCP]G and [TCP]G lesions significantly block DNA replication by Kf(-) and Dpo4, whereas [PhO]G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions.

Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

AbstractIncorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions. Primer extension reactions are commonly used for the assay of such reaction events. However, current assay protocols generally rely on radiolabeling, fluorescence reporter labeling, or removal of specific deoxynucleotide triphosphate in the reaction mixture. Herein we report on the design of two novel assay protocols that utilize a dideoxynucleotide‐terminated template strand and a phosphorothiolate‐modified deoxynucleotide‐terminated template strand. We designed and synthesized a deoxyuridine triphosphate analogue (dU*TP) containing 2‐bromoisobutyryl group and demonstrated that it could be well recognized by ϕ29DNA polymerase, E. coli DNA polymerase I Klenow Fragment, Bst DNA polymerase Large Fragment, and E. coli DNA polymerase I Klenow Fragment (exo(), which translated to effective incorporation of dU*TP into DNA. dU*TP was also successfully incorporated into extremely long single‐stranded DNA at high‐density using ϕ29 DNA polymerase by rolling circle amplification.

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity.

Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G3-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo− (Kf−) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.

Amplified Chemiluminescence Detection of DNA by Strand-Displacement Polymerization Target Recycling and G-quadruplexes/Hemin DNAzyme Catalysis

A new strategy based on strand-displacement polymerization target recycling and G-quadruplex DNAzyme catalysis was developed for amplified chemiluminescence detection of DNA. The amplified detection was achieved by using the system consisted of hairpin DNA probe, G-rich DNA, primer, and polymerase Klenow fragment exo−. When the target DNA was introduced the system, the hairpin structure of DNA probe was opened by the hybridizing of target DNA with its complementary sequence, the primer hybridized then with DNA probe and initiated polymerase-aided strand-displacement polymerization reaction, resulting in the release of target DNA and G-rich DNA. The released target DNA again hybridizes with another DNA probe to trigger the next polymerase-aided strand-displacement polymerization, generating large numbers of G-rich DNA. The G-rich DNA assembles with hemin to form the G-quadruplexes/hemin DNAzyme, which can catalyze oxidation of luminol by H2O2, generating chemiluminescence. This unique amplifying strategy gives a detection limit down to 2.5 pM, which is at least two to three orders of magnitude lower than that of unamplified DNA detection methods.