A novel biosensor based on tetrahedral DNA nanostructure and terminal deoxynucleotidyl transferase-assisted amplification strategy for fluorescence analysis of uracil-DNA glycosylase activity.

Tetrahedral DNA nanostructure (TDN), as a classical bionanomaterial, which not only has excellent structural stability and rigidity, but also possesses high programmability due to strict base-pairs complementation, is widely used in various biosensing and bioanalysis fields. In this study, we first constructed a novel biosensor based on Uracil DNA glycosylase (UDG) -triggered collapse of TDN and terminal deoxynucleotidyl transferase (TDT)-induced insertion of copper nanoparticles (CuNPs) for fluorescence and visual analysis of UDG activity. In the presence of the target enzyme UDG, the uracil base modified on the TDN were specifically identified and removed to produce an abasic site (AP site). Endonuclease IV (Endo.IV) could cleave the AP site, making the TDN collapse and generating 3'-hydroxy (3'-OH), which were then elongated under the assistance of TDT to produce poly (T) sequences. Finally, Copper (II) sulfate (Cu2+) and l-Ascorbic acid (AA) were added to form CuNPs using poly (T) sequences as templates (T-CuNPs), resulting in a strong fluorescence signal. This method exhibited good selectivity and high sensitivity with a detection limit of 8.6 × 10-5 U/mL. Moreover, the strategy has been successfully applied to the screening of UDG inhibitors and the detection of UDG activity in complex cell lysates, which means that it has promising applications in clinical diagnosis and biomedical research.

Ultrasensitive Uracil-DNA Glycosylase Activity Assay and Its Inhibitor Screening Based on Primer Remodeling Jointly via Repair Enzyme and Polymerase.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10-5 U·mL-1. Moreover, this biosensor is successfully applied for UDG inhibitor screening and complicated biological samples analysis. Compared to the previous exponential RCA methods, our proposed strategy offers additional advantages, including excellent stability, optional design of CT, and simplified operating steps. Therefore, this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.

Base excision-initiated terminal deoxynucleotide transferase-assisted amplification for simultaneous detection of multiple DNA glycosylases.

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10-3 U/ml. Moreover, it could be applied for analyzing DNA glycosylase activity in complex HeLa cell lysate samples. Therefore, this strategy possesses the advantages of high sensitivity, specificity, and multiplex, holding great potential for DNA glycosylase-related biomedical research.

Crystal Violet-Sensitized Direct Z-Scheme Heterojunction Coupled with a G-Wire Superstructure for Photoelectrochemical Sensing of Uracil-DNA Glycosylase.

Dye sensitization achieving photoelectrochemical (PEC) signal amplification for ultrasensitive bioanalysis has undergone a major breakthrough. In this proposal, an innovative PEC sensing platform is developed by combining Z-scheme WO3@SnS2 photoactive materials and a G-wire superstructure as well as a dye sensitization enhancement strategy. The newly synthesized WO3@SnS2 heterojunction with outstanding PEC performance is employed as a photoelectrode matrix. Due to the formation of the Z-scheme heterojunction between WO3 and SnS2, the migration dynamics of the photogenerated carrier is evidently augmented. To improve sensitivity, the target excision-driven dual-cycle signal amplification strategy is introduced to output exponential c-myc fragments. Crystal violet is then conjugated into the G-quadruplex to amplify the PEC signal, where crystal violet generates excited electrons by capturing visible light and rapidly injects electrons into the conduction band of SnS2, suppressing the recombination of the photo-induced carrier. Moreover, the G-wire superstructure acts as a universal amplification pathway, ensuring adequate crystal violet loads. Specifically, the biosensor for uracil-DNA glycosylase quantification displays a wide detection range (0.0005-1.0 U/mL) and a lower detection limit (0.00025 U/mL). Furthermore, the Z-scheme electron migration mechanism and the crystal violet sensitization effect are discussed in detail. The construction of the PEC sensor provides a new consideration for signal amplification and material design.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5 U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

A novel analytical principle using AP site-mediated T7 RNA polymerase transcription regulation for sensing uracil-DNA glycosylase activity.

Uracil DNA glycosylase (UDG) is a highly conserved damage repair glycosylase; the abnormal expression of DNA glycosylase has important research value in many human diseases. Therefore, highly sensitive and specific detection of UDG activity is crucial to biomedical research and clinical diagnosis. In this work, we propose an AP site-mediated T7 RNA polymerase transcription regulation analytical principle for uracil-DNA glycosylase activity analysis. T7 RNA polymerase is highly promoter-specific and only transcribes DNA downstream of the T7 promoter. We have found that modifying the T7 promoter sequence with an AP site can regulate T7 RNA polymerase transcription ability according to different modification sites. In the binding region of the promoter, AP sites greatly inhibit transcription. Moreover, AP sites in the initiation region of the promoter enhance transcription activity. Based on this research, we designed a new transcription substrate template by replacing deoxythymidine (dT) in the T7 RNA polymerase promoter sequence with one tetrahydrofuran abasic site mimic (THF) and one deoxyuridine (dU). The THF site was labeled in the transcription-enhanced region to improve transcription background, and the dU site was labeled in the transcription inhibition region to sense the UDG enzyme. In our strategy, this template can be transcribed into RNAs by T7 RNA polymerase with great multicycle amplifications. When UDG is present, dU is excised to form an AP site. The AP site damages the interaction between T7 RNA polymerase and the T7 promoter, resulting in weak transcription activity. The detection limit of this strategy is as low as 2.5 × 10-4 U mL-1, and it has good selectivity for UDG. In addition, this strategy can also detect UDG activity in complex HeLa cell lysate samples. Therefore, our developed sensor might become a promising technique for UDG activity assay.

Host-guest recognition coupled with triple signal amplification endows an electrochemiluminescent biosensor with enhanced sensitivity.

We demonstrate for the first time that host-guest recognition coupled with triple signal amplification endows an electrochemiluminescent (ECL) biosensor with enhanced sensitivity for uracil DNA glycosylase (UDG) assay. This biosensor exhibits good selectivity and extremely high sensitivity, and it can be used to screen UDG inhibitors and measure the cellular UDG activity as well.

A tri-functional probe mediated exponential amplification strategy for highly sensitive detection of Dnmt1 and UDG activities at single-cell level.

Multiplex DNA methylation and glycosylation are ubiquitous in the human body to ensure the normal function and stability of the genome. The methyltransferases and glycosylases rely on varied enzymes with different action mechanism, which still remain challenges for multiple detection. Herein, we developed a tri-functional dsDNA probe mediated exponential amplification strategy for sensitive detection of human DNA (cytosine-5) methyltransferase 1 (Dnmt1) and uracil-DNA glycosylase (UDG) activities. The tri-functional dsDNA probe was rationally designed with M-DNA and U-DNA. M-DNA contains the 5'-GCmGCGC-3' site for Dnmt1 recognition. U-DNA possesses one uracil as the substrate of UDG and a primer sequence for initiating the amplification reaction. M-DNA was complementary to partial sequence of U-DNA. In the presence of Dnmt1 and UDG, BssHⅡ and Endo Ⅳ were used to nick the 5'-GCGCGC-3' and AP sites respectively, resulting in the release of single-stranded DNA sequence (primer sequence), respectively. After magnetic separation, the released primer sequence hybridizes with padlock DNA (P-DNA), initiating exponential rolling circle amplification to produce numerous G-quadruplexes for recordable signals. The strategy exhibited the limit of detection as low as 0.009 U mL-1 and 0.003 U mL-1 for Dnmt1 and UDG, respectively. Meanwhile, this strategy was successfully applied to detect Dnmt1 and UDG activities in living cell samples at single-cell level and assay the inhibitors of Dnmt1 and UDG. Therefore, the strategy provided a potential method to detect Dnmt1 and UDG activities in biological samples for early clinic diagnosis and therapeutics.

Coupling photoelectrochemical and electrochemical strategies in one probe electrode: Toward sensitive and reliable dual-signal bioassay for uracil-DNA glycosylase activity.

Uracil-DNA glycosylase (UDG) is a typical initiator for base excision repair (BER) process. Since aberrant expression of UDG is relevant to a variety of cancers, analysis of UDG activity with high sensitivity and accuracy is of great importance. We reported herein a sensitive and reliable dual-signal bioassay for UDG activity by coupling photoelectrochemical (PEC) and electrochemical (EC) strategies in one probe electrode. The Au/TiO2 hybrid was used as a matrix to immobilize substrate DNA (sDNA), which modified with AgInS2 quantum dot (AIS QD) on the terminal. When UDG exist, the base of uracil was eliminated from the sDNA, and the produced apyrimidinic (AP) site could be cleaved by endonuclease IV (Endo. IV) immediately. Under this situation, the PEC labels of AIS QDs were detached from the electrode, resulting in a "signal-off" trend for PEC signal. After assistant DNA (aDNA) was then assembled, the hybridization chain reaction (HCR) was triggered, and EC labels of ferrocene molecules were introduced, producing a "signal-on" trend for EC signal. Besides, as the produced long double-stranded DNA by the HCR had evident steric hindrance, the PEC signal further decreased. Based on this meticulous design, the dual-signal bioassay for UDG activity showed low detection limits of 4.3 × 10-5 and 1.9 × 10-4 U/mL with PEC and EC detection, and accurate analysis of UDG activity in living cells was realized. By just changing the recognition site, this sensitive and reliable dual-signal strategy can be extended to diagnose other DNA repair-related enzymes in the real samples.

Target-triggered activation of rolling circle amplification for label-free and sensitive fluorescent uracil-DNA glycosylase activity detection and inhibition.

The expression variations of uracil-DNA glycosylase (UDG) can be used as effective biomarkers for the evaluation of gene regulation and related diseases. Here, by using a new target-triggered activation of rolling circle amplification (RCA) signal enhancement strategy, we have established a sensitive and label-free fluorescent approach for UDG activity detection and inhibition. The target UDG specifically recognizes and excises the uracil bases in a three-strand containing DNA complex to liberate one of the strands. Subsequent ligation of the excised DNA complex converts it into a suitable primer/circular template structure for the initiation of RCA for the generation of long DNA sequences with many repeated G-quadruplexes. Protoporphyrin IX further binds these G-quadruplexes to show substantially enhanced fluorescence to achieve sensitive detecting the activity of UDG with the detection limit as low as 0.00014 U mL-1. Besides, this assay approach has a high specificity toward UDG and can also be utilized to evaluate its inhibition by the uracil glycosylase inhibitor, highlighting the promising applications for convenient and sensitive UDG activity detection and inhibition for disease diagnosis and drug screening.

Electrochemiluminescent determination of the activity of uracil-DNA glycosylase: Combining nicking enzyme assisted signal amplification and catalyzed hairpin assembly.

An electrochemiluminescence (ECL) based method is described for the determination of the activity of the enzyme uracil-DNA glycosylase (UDG). It is based on the use of nicking enzyme-assisted signal amplification and catalytic hairpin assembly. UDG can recognize and hydrolyze the uracil bases from the stem of hairpin DNA1 (HP1). This causes the opening of HP1 to form a straight strand DNA. The straight HP1 can hybridize with hairpin DNA2 (HP2) to form a DNA duplex. In the presence of nicking enzyme, it can recognize and cut the specific sequences in the HP2 of the DNA duplex, and a subsequent release of HP1. It hybridizes with other HP2 to trigger the continuous cleavage of HP2, concomitantly generating abundant intermediate sequences (S1). The hairpin DNA3 (HP3) is immobilized on a gold electrode via Au-S chemistry. In the presence of S1, HP3 hybridizes with S1 and its hairpin structure is opened. This hybridization causes displacement from hairpin DNA4 (HP4), and S1 is released to initiate the next hybridization process. Thus, a massive number of HP3-HP4 duplexes is generated after the cyclic process. Subsequently, the cDNA modified on bio-bar-coded AuNP-CdSe quantum dots are immobilized on the electrode by hybridization with the redundant part of the opened HP4. This results in a significant amplification of the ECL signal. This biosensor is sensitive and selective for UDG. The detection limit is 6 mU·mL-1 and the dynamic range extends from 0.02 to 22 U·mL-1. The method was applied to real samples and gained good performance, thereby providing an ideal way for DNA repair enzyme-related biomedical research and diagnosis. Graphical abstract Schematic presentation of the electrochemiluminescence (ECL) detection of uracil-DNA glycosylase (UDG) based on nicking enzyme assisted signal amplification and catalyzed hairpin assembly. The bio-barcoded Au NP-CdSe QDs serve as the ECL signal probes to achieve a significantly signal amplification.

Determination of the activity of uracil-DNA glycosylase by using two-tailed reverse transcription PCR and gold nanoparticle-mediated silver nanocluster fluorescence: a new method for gene therapy-related enzyme detection.

The authors present a fluorometric method for ultrasensitive determination of the activity of uracil-DNA glycosylase (UDG). It is based on the use of two-tailed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and an entropy-driven reaction. The assay involves the following steps: (1) UDG-driven uracil excision repair, (2) two-tailed RT-qPCR-mediated amplification, (3) RNA polymerase-aided amplification, and (4) DNA-modified silver nanoclusters (AgNCs) as a transducer to produce a fluorescent signal. UDG enables uracil to be removed from U·A pairs in DNA1 and produces a depurinated/depyrimidinated site that is readily cleaved by endonuclease IV (Endo IV). The cleaved DNA contains the T7 RNA polymerase primer for the T7 RNA polymerase amplification which produces a large number of microRNA sequences. Subsequent two-tailed RT-qPCR leads to the formation of a prolonged DNA termed DNA3. The prolonged part of DNA3 is then hybridized with an added DNA4/DNA5 duplex, where DNA5 is labeled with gold nanoparticles (AuNPs), and DNA 4 is labeled with AgNCs. The AuNPs quench the fluorescence of the AgNCs. The duplex has a toehold to hybridize the prolong part of DNA3. This results in the formation of a DNA5/DNA3 duplex due to strand displacement (by replacing the DNA4 in the DNA4/DNA5 duplex). DNA4 is released and moves away from the AuNPs. This results in restored AgNC fluorescence, best measured at excitation/emission wavelengths of 575/635 nm. The method has a detection limit as low as 0.1 mU mL-1 of UDG activity (3σ criterion) with a range of 0.001-0.01 U mL-1. It was used to measure UDG activity in cell lysates. Conceivably, it may be used to screen for UDG inhibitors such as Ugi. Graphical abstract Schematic presentation of the two-tailed RT-qPCR assay platform for ultrasensitive detection of uracil-DNA glycosylase (UDG). Two-tailed RT-qPCR-mediated amplification and RNA polymerase-aided amplification are utilized for signal amplification. DNA-modified silver nanoclusters (AgNCs) are used as a transducer to produce a fluorescent signal.

Sensitive detection of uracil-DNA glycosylase (UDG) activity based on terminal deoxynucleotidyl transferase-assisted formation of fluorescent copper nanoclusters (CuNCs).

As an important base excision repair (BER) enzyme, uracil-DNA glycosylase (UDG) can repair the uracil-induced DNA lesion and maintain the genomic integrity. Herein, we have proposed a sensitive UDG assay based on the terminal deoxynucleotidyl transferase (TdT)-assisted formation of fluorescent copper nanoclusters (CuNCs). In this study, a uracil-containing stem-loop DNA substrate is rationally designed with its 3'-end blocked with 2', 3'-dideoxycytosine (ddC). UDG can remove the uracil in the DNA substrate to generate an apurinic/apyrimidinic (AP) site, which can then be specifically cleaved by endonuclease IV (Endo IV) to expose a 3'-OH terminus. TdT will initiate the template-free DNA extension along the exposed 3'-OH terminus to produce a quite long poly(T) tail, which will perfectly template the production of fluorescent CuNCs. By recording the fluorescence of the CuNCs, the UDG activity can be faithfully detected. In contrast, if UDG is absent, the 3'-ddC terminus of the DNA substrate cannot be recognized by TdT and thus no TdT-based extension and formation of CuNCs will occur. The use of ddC as a 3'-end blocker can greatly decrease the nonspecific DNA extension and improve the signal-to-noise ratio. Furthermore, TdT is a template-free and sequence-independent DNA polymerase, which can effectively catalyze the tailing process up to a maximum of thousands of thymines, and each tail can form many fluorescent CuNCs. Therefore, an ultrahigh sensitivity is achieved and as low as 0.00005 U/mL of UDG can be clearly detected. Moreover, with an additional AP site-contained poly(A) oligonucleotide during TdT-mediated extension, a branched amplification mechanism is also preliminarily devised, which can further push the detection limit of UDG to an extremely low level of 0.000002 U/mL.

Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase.

As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3'-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.

Integration of single-molecule detection with magnetic separation for multiplexed detection of DNA glycosylases.

We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.

Label-free and high-throughput bioluminescence detection of uracil-DNA glycosylase in cancer cells through tricyclic cascade signal amplification.

We develop a label-free and high-throughput bioluminescence method for the sensitive detection of uracil DNA glycosylase (UDG) through enzyme-mediated tricyclic cascade signal amplification. This method exhibits high sensitivity with a detection limit as low as 0.00031 U mL-1, and it can be further applied for the measurement of enzyme kinetic parameters and the screening of UDG inhibitors as well as cancer cell analysis.

Self-primer and self-template recycle rolling circle amplification strategy for sensitive detection of uracil-DNA glycosylase activity.

Sensitive and accurate detection of uracil-DNA glycosylase (UDG) activity is available for evaluating and validating their function in uracil base-excision repair (UBER) pathway and clinical diagnosis. Here, a sensitive and accurate method for UDG activity detection was developed on the basis of self-primer and self-template recycle rolling circle amplification (Self-RRCA) strategy. First, an immature template (IT) with a uracil base and an Nt.BbvCI nicking site was designed, which could hybridize with a designed primer to form a pre-amplicon probe (PA probe). Under the action of UDG, the uracil base in the PA probe could be removed to generate an apyrimidinic (AP) site. Then the generated AP site was excised by endonuclease IV (endo IV), making the PA probe form a RCA amplicon through reconformation. The RCA amplicon subsequently was used to trigger the RCA, and after Nt.BbvCI nicking reaction, new amplicons were released to initiate next RCA, constituting a Self-RRCA. In this method, the designed IT was not fully complementary with the primer in the ligation part, which could effectively avoid nonspecific ligation reaction and eventually effectively avoid nonspecific amplification. Compared with the linear RCA, the Self-RRCA exhibited higher amplification efficiency. Due to above advantages, a sensitive and accurate detection method was achieved with a limit of 4.68 × 10-5 U mL-1. Furthermore, the method was adopted to screen the inhibitor of UDG and assay the activity of UDG in HeLa cell lysate. This method will offer a promising analysis tool for further biomedical research of UDG and clinical diagnosis.

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.

A C-HCR assembly of branched DNA nanostructures for amplified uracil-DNA glycosylase assays.

An autonomous nonenzymatic DNA machine has been successfully engineered based on a two-layered cascaded hybridization chain reaction (C-HCR) circuit, in which the tandem outputs of the upstream HCR-1 unit activate the downstream HCR-2 unit to induce successive repeated hybridizations, generating branched DNA structures and enabling sensitive and selective detection of uracil-DNA glycosylase and its inhibitors.

Aptamer-mediated universal enzyme assay based on target-triggered DNA polymerase activity.

We herein describe an innovative method for a universal fluorescence turn-on enzyme assay, which relies on the target enzyme-triggered DNA polymerase activity. In the first target recognition step, the target enzyme is designed to destabilize detection probe derived from an aptamer specific to DNA polymerase containing the overhang sequence and the complementary blocker DNA, which consequently leads to the recovery of DNA polymerase activity inhibited by the detection probe. This target-triggered polymerase activity is monitored in the second signal transduction step based on primer extension reaction coupled with TaqMan probe. Utilizing this design principle, we have successfully detected the activities of two model enzymes, exonuclease I and uracil DNA glycosylase with high sensitivity and selectivity. Since this strategy is composed of separated target recognition and signal transduction modules, it could be universally employed for the sensitive determination of numerous different target enzymes by simply redesigning the overhang sequence of detection probe, while keeping TaqMan probe-based signal transduction module as a universal signaling tool.