Presence Of Uracil DNA Glycosylase Research Articles

ATP-driven AP endonuclease-active Exo III@zeolitic imidazolate framework for uracil-DNA glycosylase imaging in living cells

In the currently available approaches for intracellular uracil-DNA glycosylase (UDG) detection, their signal outputs usually rely on the cleavage of the residual AP sites in DNA probes by apurinic/apyrimidinic endonuclease 1 (APE1), whose expression level varies across different types of cells. Therefore, reliable visualizing detection of UDG in living cells remains challenging. Herein, a nanoprobe for imaging of intracellular UDG activity has been designed by taking advantages of both zeolitic imidazolate framework-90 (ZIF-90) and endonuclease III. ZIF-90 is used to deliver exonuclease III (Exo III) and hairpin DNA strands into cells. One of the hairpin strands (HP1) contains a deoxyuridine (dU) base in its stem region, while the other (HP2) was modified with FAM molecule and BHQ1 at its 3′ and 5′ ends, respectively. In the presence of UDG, the dU base in HP1 is excised to form an AP site. Exo III can cleave the AP site in HP1 to generate a stem-loop fragment, which can hybridize with HP2 to initiate the recycling digestion of HP2 by Exo III, resulting in amplification of the fluorescence signal and hence sensitive detection of UDG (limit of detection: 2.0×10−6 U/mL). Experimental results have demonstrated that the nanoprobe has merits of good biocompatibility, high sensitivity and selectivity. It realizes in-situ imaging of intracellular UDG without the involvement of APE1, is a promising alternative tool for UDG detection in fields of clinical diagnosis and biomedical research.

Distance-based paper device coupled with uracil-rich DNA hydrogel for visual quantification of Uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG), an enzyme for repairing uracil-containing DNA damage, is crucial for maintaining genomic stability. Simple and fast quantification of UDG activity is essential for biological assay and clinical diagnosis, since its aberrant level is associated with DNA damage and various diseases. Herein, we developed a fully integrated “sample in-signal out” distance-based paper analytical device (dPAD) for visual quantification of UDG using a flow-controlled uracil-rich DNA hydrogel (URDH). The uracil base sites contained in the DNA hydrogel are mis-incorporated with dUTP by rolling circle amplification (RCA), which simplifies the preparation process of the functionalized hydrogel. In the presence of UDG, the uracil in URDH can be recognized and removed to induce the permeability change of URDH, resulting in the visible distance signal along the paper channel. Using dPAD, as low as 6.4 × 10−4 U/mL of UDG (within 80 min) is visually identified without any instruments and complicated operations. This integrated dPAD is advantageous for its simplicity, cost effectiveness, and ease of use. We envision that it has the great potential for point-of-care testing (POCT) in DNA damage testing, personalized healthcare assessment, and biomedical applications.

An enzyme-free electrochemical biosensor based on NiCoP@PtCu nanozyme and multi-MNAzyme junctions for ultrasensitive Uracil-DNA glycosylase detection

Abnormal expressions of uracil-DNA glycosylase (UDG) have been associated with various diseases. We proposed a novel electrochemical biosensing platform-based strategy for UDG detection. This method combined NiCoP@PtCu nanozyme with multi-MNAzyme junctions. The U base was deleted in the presence of UDG, resulting in self-assembly of multi-MNAzyme junctions with the ability to continually cleave DNA hairpins and release various products. The products could bind the protective probes, exposing capture probes on the electrode’s surface. Moreover, the exposed probes hybridized with the sDNAs-labeled NiCoP@PtCu nanozyme, yielding a significant electrochemical signal. The established biosensor was enzyme-free and with an ultrahigh sensitivity. Benefiting from the twofold amplification of multi-MNAzyme junctions and nanozyme, the sensing technique exhibited a large dynamic range, from 0.001 to 1.0 U mL −1 and a low detection of 6.6 × 10−4 U mL −1. In addition, the method was effectively used for UDG detection in lysed cells. Therefore, the biosensor we established has broad prospects in clinical diagnosis and biomedical research.

Sensitive electrochemical biosensor for Uracil-DNA glycosylase detection based on self-linkable hollow Mn/Ni layered doubled hydroxides as oxidase-like nanozyme for cascade signal amplification

Nanozymes have been widely used in biosensors instead of natural enzymes because of low cost, high stability and ease of storage. However, few works use oxidase-like nanozymes to fabricate electrochemical biosensors. Herein, we proposed a sensitive electrochemical biosensor to detect uracil-DNA glycosylase (UDG) based on the hollow Mn/Ni layered doubled hydroxides (h-Mn/Ni LDHs) as oxidase-like nanozyme. Briefly, the h-Mn/Ni LDHs, which was prepared by a facile hydrothermal method, exhibited excellent oxidase-like activity because the hollow structure provided rich active sites and high specific surface area. Then, the signal probes were constructed by assembling the hairpin DNA (hDNA), single DNA1 and DNA2 on the h-Mn/Ni LDHs, respectively. In the presence of UDG, the uracil bases in the stem of hDNA were specifically excised, generating apyrimidinic (AP) sites and inducing the unwinding of the hDNA. Afterwards, the h-Mn/Ni LDHs@Au-hDNA/DNA1 was connected on the electrode via hybridization between unwinded hDNA and capture DNA (cDNA). Subsequently, the self-linking process allowed the retention of numerous h-Mn/Ni LDHs through simple DNA hybridization to amplify the signal of o-phenylenediamine (o-PD). Unlike many peroxidase-like nanozyme-based electrochemical biosensors, there is no need to add H2O2 during the experimental process, which effectively reduced the background signal as well as improved the stability of the biosensor. As expected, the biosensor exhibited excellent performance with a wide linear range and a low detection limit. This work highlights an appealing opportunity to develop a no H2O2 platform based on h-Mn/Ni LDHs for future application in biological analysis and clinical diagnosis.

Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level.

DNA repair enzymes (e.g., DNA glycosylases) play a critical role in the repair of DNA lesions, and their aberrant levels are associated with various diseases. Herein, we develop a sensitive method for simultaneous detection of multiple DNA repair enzymes based on the integration of single-molecule detection with rolling circle amplification (RCA)-driven encoding of different fluorescent molecules. We use human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) as the target analytes. We design a bifunctional double-stranded DNA (dsDNA) substrate with a hypoxanthine base (I) in one strand for hAAG recognition and an uracil (U) base in the other strand for UDG recognition, whose cleavage by APE1 generates two corresponding primers. The resultant two primers can hybridize with their respective circular templates to initiate RCA, resulting in the incorporation of multiple Cy3-dCTP and Cy5-dGTP nucleotides into the amplified products. After magnetic separation and exonuclease cleavage, the Cy3 and Cy5 fluorescent molecules in the amplified products are released into the solution and subsequently quantified by total internal reflection fluorescence (TIRF)-based single-molecule detection, with Cy3 indicating the presence of hAAG and Cy5 indicating the presence of UDG. This strategy greatly increases the number of fluorescent molecules per concatemer through the introduction of RCA-driven encoding of different fluorescent molecules, without the requirement of any specially labeled detection probes for simultaneous detection. Due to the high amplification efficiency of RCA and the high signal-to-ratio of single-molecule detection, this method can achieve a detection limit of 6.10 × 10-9 U mL-1 for hAAG and 1.54 × 10-9 U mL-1 for UDG. It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and the screening of DNA glycosylase inhibitors, holding great potential in early clinical diagnosis and drug discovery.

Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification.

Sensitive detection of uracil-DNA glycosylase (UDG) activity is very important in the study of many fundamental biochemical processes and clinical applications. Here, we develop a novel assay for the detection of UDG activity by using the self-initiating multiple rolling circle amplification (SM-RCA) strategy. We first design a trigger probe modified with NH2 at its 3′-terminal and uracil base in the middle of sequence, which is complementary to a cyclized padlock probe. In the presence of UDG, a uracil base can be excised by UDG to generate an apurinic/apyrimidinic (AP) site. The AP site is recognized and cleaved by endonuclease IV (Endo IV), releasing the primer with 3′-OH. The primer can trigger the rolling circle amplification (RCA) reaction, producing a long and repeated DNA strand embedded some uracil bases. These uracil bases can be cleaved by UDG and Endo IV again, and then, more primers are generated to initiate SM-RCA reaction, producing large amounts of DNA product. Afterward, the DNA product is measured by a specific DNA fluorescence dye for quantitative detection of UDG activity. The linear range of the method is 5 × 10–5 to 1.25 × 10–3 U/mL, and the detection limit is 1.7 × 10–5 U/mL. This method not only utilizes the target UDG itself to trigger RCA but also further induces SM-RCA reaction, providing a simple, sensitive, and cost-effective strategy for the detection of glycosylase and clinical diagnosis.

Label-free and sensitive detection of uracil-DNA glycosylase using exponential real-time rolling circle amplification

Schematic representation of an exponential rolling circle amplification-based uracil-DNA glycosylase activity-sensing platform.

Abasic Site-Assisted Inhibition of Nicking Endonuclease Activity for the Sensitive Determination of Uracil DNA Glycosylase.

We herein describe A novel strategy to accurately determine uracil DNA glycosylase (UDG) activity is described based on the finding that nicking endonuclease-assisted cleavage reaction can be regulated by the presence of abasic site. This strategy utilizes DNA probes rationally designed to contain uracil base at the cleavage site for nicking endonuclease, which is coupled to the isothermal nicking endonuclease amplification reaction (NEAR) method. In the absence of UDG, intact DNA probes generate a large number of double-stranded (ds) DNA products through the NEAR, but the presence of UDG that converts uracil base into abasic site suppresses nicking endonuclease activity and the subsequent NEAR. As a result, dsDNA products are not produced, which is simply monitored by the dsDNA specific fluorescence dye, SYBR green I. By employing this strategy, we sensitively determined the UDG activity down to 0.003 U mL-1 with high specificity over other base excision enzymes. In addition, the diagnostic capability of this method was successfully verified by reliably assaying UDG present in a human serum sample.

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1−/− mice. We found that 5-hydroxymethyluracil accumulated in Smug1−/− tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1−/− mice did not accumulate uracil in their genome and Ung−/− mice showed slightly elevated uracil levels. Contrastingly, Ung−/−Smug1−/− mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation

We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research.

A novel and label-free biosensors for uracil-DNA glycosylase activity based on the electrochemical oxidation of guanine bases at the graphene modified electrode

Uracil-DNA glycosylase (UDG) as an important base excision repair enzymes is widely distributed in organism, and it plays a crucial role in sustaining the genome integrity. Therefore, it is significant to carry out the analysis of UDG activity. In this present work, a novel and label-free electrochemical sensing platform for the sensitive detection of uracil DNA glycosylase (UDG) activity has been developed. Herein, the graphene modified glassy carbon (GC) electrode was prepared. And two complementary DNA strands were hybridized to form dsDNA (P1P2). In the presence of UDG, the uracil bases in P1P2 were specifically hydrolyzed, inducing the unwinding of the DNA duplex, and accompanied by the release of P1. Thus, the released P1 was adsorbed onto the graphene/GC electrode surface via π–π stacking. By investigating the electrochemical behavior of P1 at the graphene/GC electrode, the electrochemical oxidation of guanine bases in P1 was obviously observed. Therefore, using the current responses of guanine base in P1 as a signal indicator, UDG activity can be simply determined with high sensitivity, and the detectable lowest concentration is 0.01U/mL. This present design does not need covalent attachment of redox indicator to DNA, preventing participation of redox labels in the background. Meanwhile, the proposed strategy for the assay of UDG activity also has a remarkable sensitivity due to the excellent properties of graphene, which could increase both the immobilization amount of released ssDNA and the conductivity of the sensing system. All these elucidate that this developed protocol may lay a potential foundation for the sensitive detection of UDG activity in clinical diagnosis.

Label-free colorimetric assay for base excision repair enzyme activity based on nicking enzyme assisted signal amplification

Specific and sensitive detection of base excision repair enzyme activity is essential to many fundamental biochemical process researches. Here, we propose a novel label-free homogeneous strategy for visualized uracil DNA glycosylase (UDG) activity assay based on nicking enzyme assisted signal amplification. In this method two hairpin probes were employed for the colorimetric detection, namely hairpin probe 1 (HP 1) carrying two uracil residues in the stem, and hairpin probe 2 (HP 2) containing a G-riched DNAzyme segment, and the recognition sequence as well as the cleavage site for the nicking enzyme. In the presence of UDG, the uracil bases in the stem of HP 1 can be specifically recognized and hydrolyzed by UDG, which leads to the destabilization of its stem containing abasic sites (AP sites), and then results in the opening of HP 1 to form a single strand. The opened HP 1 hybridizes with HP 2 to form a DNA duplex, which initiates the specific cleavage of HP 2 by the nicking enzyme, leading to the release of G-riched DNAzyme segments. As a result, HP 1 is released and able to hybridize with another HP 2 to induce the continuous cleavage of HP 2, generating enormous amount of G-riched DNAzyme segments. Finally, the G-riched DNAzyme segments bind hemin to form a catalytically active G-quadruplex–hemin DNAzyme which can catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the colored ABTS–, providing a visible signal for UDG activity detection. This assay exhibits several advantages such as simplicity, low-cost, high selectivity and desirable sensitivity, which shows great potential of providing a promising platform for convenient and visualized analysis of UDG or other biomolecules.

A novel electrochemical biosensor for label-free detection of uracil DNA glycosylase activity based on enzyme-catalyzed removal of uracil bases inducing strand release

A novel and label-free electrochemical sensing platform for sensitive detection of uracil DNA glycosylase (UDG) activity has been developed, which is based on UDG-catalyzed removal of uracil bases inducing strand release with methylene blue (MB) as an electrochemical hybridization indicator. The capture DNA containing a thiol group was first immobilized on the gold electrode whose surface was pre-modified with dendritic gold nanoparticles by direct electrodeposition, followed by hybridization with its complementary single-strand DNA containing four uracil bases located in the intermediate positions. Subsequently, MB was abundantly intercalated into the formed DNA duplex structure, resulting in a measurable electrochemical signal. In the presence of UDG, the uracil bases were specifically hydrolyzed, which could induce the unwinding of the DNA duplex, accompanied by the release of the intercalated MB from the electrode surface, leading to the corresponding proportional reduction of the redox signal. In the present work, the proposed sensing platform exhibits a good linear response to the logarithm of UDG concentration in the range from 0.025 to 2 U mL−1 with a detection limit of 0.012 U mL−1. In addition, the proposed strategy shows advantages of desirable sensitivity, high selectivity and low cost. It is the first example that employs the electrochemical assay for the detection of UDG activity. Therefore, this novel method will demonstrate promising prospects in a wide range of applications in the future, such as in-depth researches of UDG and clinical diagnosis.

The catalytic power of uracil DNA glycosylase in the opening of thymine base pairs.

Uracil DNA glycosylase (UNG) locates uracil and its structural congener thymine in the context of duplex DNA using a base flipping mechanism. NMR imino proton exchange measurements were performed on free and UNG-bound DNA duplexes in which a single thymine (T) was paired with a series of adenine analogues (X) capable of forming one, two, or three hydrogen bonds. The base pair opening equilibrium for the free DNA increased 55-fold as the number of hydrogen bonds decreased, but the opening rate constants were nearly the same in the absence and presence of UNG. In contrast, UNG was found to slow the base pair closing rate constants (kcl) compared to each free duplex by a factor of 3- to 23-fold. These findings indicate that regardless of the inherent thermodynamic stability of the TX pair, UNG does not alter the spontaneous opening rate. Instead, the enzyme holds the spontaneously expelled thymine (or uracil) in a transient extrahelical sieving site where it may partition forward into the enzyme active site (uracil) or back into the DNA base stack (thymine).

Enzymatic repair of specific oxidative damages to DNA deoxyribose in [formula omitted

Specific and sensitive detection of base excision repair enzyme activity is essential to many fundamental biochemical process researches. Here, we propose a novel label-free homogeneous strategy for visualized uracil DNA glycosylase (UDG) activity assay based on nicking enzyme assisted signal amplification. In this method two hairpin probes were employed for the colorimetric detection, namely hairpin probe 1 (HP 1) carrying two uracil residues in the stem, and hairpin probe 2 (HP 2) containing a G-riched DNAzyme segment, and the recognition sequence as well as the cleavage site for the nicking enzyme. In the presence of UDG, the uracil bases in the stem of HP 1 can be specifically recognized and hydrolyzed by UDG, which leads to the destabilization of its stem containing abasic sites (AP sites), and then results in the opening of HP 1 to form a single strand. The opened HP 1 hybridizes with HP 2 to form a DNA duplex, which initiates the specific cleavage of HP 2 by the nicking enzyme, leading to the release of G-riched DNAzyme segments. As a result, HP 1 is released and able to hybridize with another HP 2 to induce the continuous cleavage of HP 2, generating enormous amount of G-riched DNAzyme segments. Finally, the G-riched DNAzyme segments bind hemin to form a catalytically active G-quadruplex–hemin DNAzyme which can catalyze the H2O2-mediated oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2−) to the colored ABTS–, providing a visible signal for UDG activity detection. This assay exhibits several advantages such as simplicity, low-cost, high selectivity and desirable sensitivity, which shows great potential of providing a promising platform for convenient and visualized analysis of UDG or other biomolecules.