Endonuclease IV and T4 ligase enhanced detection of mutations in low abundance.

We propose a mutant detection approach based on endonuclease IV and DNA ligase in combination with qPCR. The enzymes functioned cooperatively to facilitate PCR for low abundance DNA detection. We demonstrate that our approach can distinguish mutations as low as 0.01%, indicating the potential application of this strategy in early cancer diagnosis.

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.

Cloning and characterization of the major AP endonuclease from Staphylococcus aureus.

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Complete genome analysis of Staphylococcus aureus identified a single AP endonuclease, SaNfo, which is a member of the endonuclease IV family exemplified by Escherichia coli Nfo. At present, it remains unknown whether SaNfo possesses DNA repair activities similar to its counterparts from E. coli and other bacteria. Here, we report that the purified SaNfo protein contains efficient AP endonuclease and nucleotide incision repair (NIR) activities. Optimal reaction conditions for SaNfo-catalysed AP endonuclease activity are high ionic strength and Mn2+ concentration, pH in range 7.5-9.0 and the temperature optimum of 37-45 °C. Cell-free extracts of S. aureus exhibited efficient AP site cleavage and NIR activities. Heterologous expression of SaNfo strongly reduces the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2. Site-directed mutagenesis showed that the Glu258 residue is critical for the SaNfo enzyme function. The AP endonuclease but not the NIR activity of SaNfo were stimulated by the ß-clamp (SaDnaN dimer), suggesting that it might participate in the organization of BER in S. aureus. Overall, our data confirm that the activity, substrate specificity and in vivo functionality of S. aureus Nfo are consistent with this protein being the major AP endonuclease for the repair of DNA damage generated by endogenous and host-imposed factors.

Sensitive detection of abasic sites in double-stranded DNA based on the selective reaction of enzymes.

The apurinic/apyrimidinic (AP) site is one of the most common DNA lesions and a critical intermediate during the base excision repair pathway. Therefore, AP sites are essential in clinical diagnosis, treatment and detection. However, the existing detection methods are complicated in design and synthesis and have high instrument requirements, limiting their wide application. Therefore, there is an urgent need for a sensitive and straightforward detection method without time-consuming and heterogeneous reactions. Herein, we developed two compatible detection methods for AP sites in long and short dsDNA. For long and short dsDNA, the background signal was successfully suppressed by the affinity difference of Terminal deoxynucleotidyl transferase (TdT) and 3' -end blocking, respectively, thus achieving high detectability and specificity. The detection limit was 13 pM in 20 µL, meaning that the LOD was 0.26 fmol for AP site amount and 0.05% for AP site abundance. The method has been successfully applied to detect AP sites in various biological samples quickly. Therefore, it has broad clinical application prospects, catering for the need for a point of care.

In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5'-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.

A universal probe system for low-abundance point mutation detection based on endonuclease IV.

Single base mutations are closely related to cancer diagnosis and treatment. The fluorescent probe method is one of the important methods to detect single-base mutations. We constructed a universal probe detection system based on endonuclease IV and the DNA strand displacement reaction. The system uses two toehold strand displacement reactions to relay the mutation information to the universal strand. There is no need to design the probe one-by-one for each mutation point during multi-site detection. It has the advantages of simple operation, rapid detection, and low cost. We used this method to detect common clinical mutation sites (PTEN R130Q/EGFR L858R/PTEN rs1473918395), and the detection limit can reach 0.1%-1%. The detection system can provide a new rapid and economical method for clinical single-base mutation detection, and has broad application prospects in diagnosis and prognostic evaluation.

Endonuclease IV-Regulated DNAzyme Motor for Universal Single-nucleotide Variation Discrimination.

Single-nucleotide variation (SNV) detection plays significant roles in disease diagnosis and treatment. Generally, auxiliary probe, restricted design rules, complicated detection system, and repeated experimental parameter optimization are needed to obtain satisfactory tradeoff between sensitivity and selectivity for SNV discrimination, especially when different mutant sites need to be distinguished. To overcome these limitations, we developed a universal, straightforward, and relatively cheap SNV discrimination strategy, which simultaneously possessed high sensitivity and selectivity. The excellent performance of this strategy was ascribed to the SNV discrimination property of endonuclease IV (Endo IV) and the different hydrolysis behavior between free deoxyribozyme (DNAzyme) and the trapped DNAzyme to the substrates modified on gold nanoparticles (AuNPs). When Endo IV recognized the mutant-type target (MT), free DNAzyme was released from the probe, and the DNAzyme motor was activated with the help of cofactor Mn2+ to generate an amplified fluorescence signal. On the contrary, the wild-type target (WT) could not effectively trigger the DNAzyme motor. Moreover, for different SNV types, the corresponding probe could be designed by simply changing the sequence hybridized with the target and retaining the DNAzyme sequence. Thus, the fluorescence signal generation system does not need to change for different SNV targets. Five clinical-related SNVs were determined with the limit of detection (LOD) ranging from 0.01 to 0.05%, which exhibited competitive sensitivity over existing SNV detection methods. This strategy provided another insight into the properties of Endo IV and DNAzyme, expanded the applications of DNAzyme motor, and has great potential to be used for precision medicine.

[Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis].

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection.

Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.

Integration of single-molecule detection with endonuclease IV-assisted signal amplification for sensitive DNA methylation assay.

We demonstrate the development of a new fluorescent biosensor for sensitive DNA methylation assay by integrating single-molecule detection with endo IV-assisted signal amplification. This biosensor possesses the characteristics of good selectivity and high sensitivity with a detection limit of 7.3 × 10-17 M. It can distinguish as low as 0.01% methylation level, and can analyze genomic DNA methylation even in a single cancer cell.

Determination of low-abundance single-base point mutations based on endonuclease IV and branch migration system.

Combining endonuclease IV and branch migration competition systems, we have designed a new single-base mutation detection method which is capable of thermostatic, sensitive, simple, and cost-effective at the same time, while other probe method based on endonuclease IV hardly achieve. Our method has better discrimination factors (6.81-83.10) and a low abundance detection limit of mutant-type DNA (MT) (0.05% mutant-type DNA/total DNA) without complex temperature optimize process. The concentration detection limit of MT is 0.03 nM.The abundance detection limit for EGFR L858R (0.1%) and PTEN R130Q (0.05%) in clinical samples suggested that the method has potential applications in clinical diagnosis.

Development of a bidirectional isothermal amplification strategy for the sensitive detection of transcription factors in cancer cells.

We developed a new strategy to sensitively detect transcription factors (TFs) based on the integration of a bidirectional isothermal exponential amplification reaction (EXPAR) with endonuclease IV (endo IV)-assisted cycle digestion of signal probes. This assay exhibits ultrahigh sensitivity with a detection limit of 1.29 × 10-14 M, and it can measure endogenous NF-κB p50 in HeLa cell extracts. Moreover, this strategy can be applied to screen TF inhibitors and detect other TFs by simply changing the TF-binding sequence.

Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA walker and hybridization chain reaction amplification.

An ultrasensitive fluorescence sensing strategy for kanamycin (KANA) determination using endonuclease IV (Endo IV)-powered DNA walker, and hybridization chain reaction (HCR) amplification was reported. The sensing system consists of Endo IV-powered 3D DNA walker using for the specific recognition of KANA and the formation of the initiators, two metastable hairpin probes as the substrates of HCR and a tetrahydrofuran abasic site (AP site)-embeded fluorescence-quenched probe for fluorescence signal output. On account of this skilled design of sensing system, the specific binding between KANA and its aptamer activates DNA walker, in which the swing arm can move autonomously along the 3D track via Endo IV-mediated hydrolysis of the anchorages, inducing the formation of initiators that initiates HCR and the following Endo IV-assisted cyclic cleavage of fluorescence reporter probes. The use of Endo IV offers the advantages of simplified and accessible design without the need of specific sequence in DNA substrates. Under the optimal experimental conditions, the fluorescence biosensor shows excellent sensitivity toward KANA detection with a detection limit as low as 1.01 pM (the excitation wavelength is 486 nm). The practical applicability of this strategy is demonstrated by detecting KANA in spiked milk samples with recovery in the range of 98 to 102%. Therefore, this reported strategy might create an accurate and robust fluorescence sensing platform for trace amounts of antibiotic residues determination and related safety analysis. Graphical abstract Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA Walker and hybridization chain, reaction amplification, Xiaonan Qu, Jingfeng Wang, Rufeng Zhang, Yihan Zhao, Shasha Li, Yu Wang, Su Liu*, Jiadong Huang, and Jinghua Yu, an ultrasensitive fluorescence sensing strategy for kanamycin determination using endonuclease IV-powered DNA walker, and hybridization chain reaction amplification is reported.

Proximity-enabled bidirectional enzymatic repairing amplification for ultrasensitive fluorescence sensing of adenosine triphosphate.

A novel fluorescence sensing strategy for ultrasensitive and highly specific detection of adenosine triphosphate (ATP) has been developed by the combination of the proximity ligation assay with bidirectional enzymatic repairing amplification (BERA). The strategy relies on proximity binding-triggered the release of palindromic tail that initiates bidirectional cyclic enzymatic repairing amplification reaction with the aid of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). A fluorescence-quenched hairpin probe with a palindromic tail at the 3' end is skillfully designed that functions as not only the recognition element, primer, and polymerization template for BERA but also the indicator for fluorescence signal output. On the basis of the amplification strategy, this biosensor displays excellent sensitivity and selectivity for ATP detection with an outstanding detection limit of 0.81 pM. Through simultaneously enhancing the target response signal value and reducing nonspecific background, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, our biosensor also shows promising potential in real sample analysis. Therefore, the proximity-enabled BERA strategy indeed creates a simple and valuable fluorescence sensing platform for ATP identification and related disease diagnosis and biomedical research.

Branch migration-based polymerase chain reaction combined with endonuclease IV-assisted target recycling probe/blocker system for detection of low-abundance point mutations.

Sensitive detection of low-abundance point mutations in blood or tissue may provide a great opportunity for the minimally invasive diagnosis of cancer and other related diseases. We demonstrate a novel method for ultra-sensitive detection of point mutations at low abundance by combination of branch migration-based PCR with endonuclease IV-assisted target recycling probe/blocker system. The method is able to identify the point mutations at abundances down to 0.01-0.02%. We anticipate this method to be widely adopted in clinical diagnosis and molecular research.

Depurination of Colibactin-Derived Interstrand Cross-Links.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

Properties and efficient scrap-and-build repairing of mechanically sheared 3' DNA ends.

Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3' termini (about 20% of the total) was not repaired after the removal of 3' phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3'- > 5' exonuclease activity of exonuclease III (3'- > 5' exonuclease, insensitive to the 3' phosphate group) was counterbalanced by the 5'- > 3' polymerase activity of T4DP. This combined reaction, termed "SB-repairing" (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.

Eliminating the secondary structure of targeting strands for enhancement of DNA probe based low-abundance point mutation detection.

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.

Primer remodeling amplification-activated multisite-catalytic hairpin assembly enabling the concurrent formation of Y-shaped DNA nanotorches for the fluorescence assay of ochratoxin A.

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.

Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase.

DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies. Thus, the detection of DG activity is essential for biomedical research and clinical diagnosis. Herein, we develop a facile fluorometric method based on the base excision repair (BER) mediated cascading triple-signal amplification for the sensitive detection of DG. The presence of human alkyladenine DNA glycosylase (hAAG) can initiate the cleavage of the substrate at the mismatched deoxyinosine site by endonuclease IV (Endo IV), resulting in the breaking of the DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate strand displacement amplification (SDA) to release primers. The released primers can further bind to a circular template to induce an exponential primer generation rolling circle amplification (PG-RCA) reaction, producing a large number of primers. The primers that resulted from the SDA and PG-RCA reaction can induce the subsequent recycling cleavage of signal probes, leading to the generation of a fluorescence signal. Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification, this method exhibits a low detection limit of 0.026 U mL-1 and a large dynamic range of 4 orders of magnitude for hAAG. Moreover, this method has distinct advantages of simplicity and low cost, and it can further quantify the hAAG activity from HeLa cell extracts, holding great potential in clinical diagnosis and biomedical research.