uracil-DNA Glycosylase Inhibitor Research Articles

Single G-quadruplex-based fluorescence method for the uracil-DNA glycosylase inhibitor screening

Uracil-DNA glycosylase (UDG) plays a pivotal role in the base repair system. Through bioinformatics, we found that the expression of the UDG enzyme in many cancer cells is increased, and its high expression is not conducive to the prognosis of lung cancer patients. The development of analytical techniques for the quantification of UDG activity and the identification of UDG inhibitors is of paramount importance. We found that when the T base in the G4 loop region mutated to uracil, the G4 structure was not disrupted and still retained the characteristics of a G4 structure (emitting strong fluorescence after binding with ThT (Thioflavin T). Inspired by this phenomenon, we developed a detection method for UDG and its inhibitors utilizing a single DNA strand engineered to form a G-quadruplex structure, containing uracil residues within the loop region, designated as G4-dU. The inclusion of uracil-DNA glycosylase (UDG) in the assay environment induces the removal of uracil, resulting in the formation of apurinic sites (AP) within the G4-dU sequence. Subsequent thermal denaturation leads to strand cleavage at AP sites, precluding the reformation of the G-quadruplex configuration and abrogating fluorescence emission. The detection process in this study can be completed in only 30 min to 1 h, offers a straightforward, expedient, and efficacious modality for assessing UDG activity and UDG inhibitor potency, thereby facilitating the discovery of novel therapeutic agents for cancer treatment.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

Systems metabolic engineering of Escherichia coli for high-yield production of Para-hydroxybenzoic acid

Para-hydroxybenzoic acid (PHBA) is extensively used as an additive in the food and cosmetics industries, significantly enhancing product shelf life and stability. While microbial fermentation offers an environment-friendly and sustainable method for producing PHBA, the titer and productivity are limited due to product toxicity and complex metabolic flux distributions. Here, we initially redesigned a L-phenylalanine-producing Escherichia coli by employing rational metabolic engineering strategies, resulting in the production of PHBA reached the highest reported level of 14.17 g/L. Subsequently, a novel accelerated evolution system was devised comprising deaminase, the alpha subunit of RNA polymerase, an uracil-DNA glycosylase inhibitor, and the PHBA-responsive promoter PyhcN. This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC50) for PHBA within 15 days. Finally, the evolved strain achieved a production of 21.35 g/L PHBA in a 5-L fermenter, with a yield of 0.19 g/g glucose and a productivity rate of 0.44 g/L/h. This engineered strain emerges as a promising candidate for industrial production of PHBA through an eco-friendly approach.

DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay

BackgroundAs a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. ResultsWe herein describe a UDG-REtarded CRISPR Amplification assay, termed ‘URECA’. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10−4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SignificanceURECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

Detection of Uracil-Excising DNA Glycosylases in Cancer Cell Samples Using a Three-Dimensional DNAzyme Walker.

DNA glycosylase dysregulation is implicated in carcinogenesis and therapeutic resistance of cancers. Thus, various DNA-based detection platforms have been developed by leveraging the base excision activity of DNA glycosylases. However, the efficacy of DNA-based methods is hampered due to nonspecific degradation by nucleases commonly present in cancer cells and during preparations of cell lysates. In this report, we describe a fluorescence-based assay using a specific and nuclease-resistant three-dimensional DNAzyme walker to investigate the activity of DNA glycosylases from cancer cell lysates. We focus on DNA glycosylases that excise uracil from deoxyuridine (dU) lesions, namely, uracil DNA glycosylase (UDG) and single-stranded monofunctional uracil DNA glycosylase (SMUG1). The limits of detection for detecting UDG and SMUG1 in the buffer were 3.2 and 3.0 pM, respectively. The DNAzyme walker detected uracil excision activity in diluted cancer cell lysate from as few as 48 A549 cells. The results of the UDG inhibitor experiments demonstrate that UDG is the predominant uracil-excising glycosylase in A549 cells. Approximately 500 nM of UDG is present in each A549 cell on average. No fluorescence was generated in the samples lacking DNAzyme activation, indicating that there was no nonspecific nuclease interference. The ability of the DNAzyme walker to respond to glycosylase activity illustrates the potential use of DNAzyme walker technology to monitor and study biochemical processes involving glycosylases.

Repurposing sodium stibogluconate as an uracil DNA glycosylase inhibitor against prostate cancer using a time-resolved oligonucleotide-based drug screening platform

Repurposing drugs can significantly reduce the time and costs associated with drug discovery and development. However, many drug compounds possess intrinsic fluorescence, resulting in aberrations such as auto-fluorescence, scattering and quenching, in fluorescent high-throughput screening assays. To overcome these drawbacks, time-resolved technologies have received increasing attention. In this study, we have developed a rapid and efficient screening platform based on time-resolved emission spectroscopy in order to screen for inhibitors of the DNA repair enzyme, uracil-DNA glycosylase (UDG). From a database of 1456 FDA/EMA-approved drugs, sodium stibogluconate was discovered as a potent UDG inhibitor. This compound showed synergistic cytotoxicity against 5-fluorouracil-resistant cancer cells. This work provides a promising future for time-resolved technologies for high-throughput screening (HTS), allowing for the swift identification of bioactive compounds from previously overlooked scaffolds due to their inherent fluorescence properties.

Cytosine base editors optimized for genome editing in potato protoplasts.

In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3-4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.

Colorimetric and electrochemical dual-signal detection of uracil-DNA glycosylase using functionalized pure DNA hydrogel on paper-based analytical devices

The development of simple and accurate detection of uracil-DNA glycosylase (UDG) is of great significance for early clinical diagnosis and biomedical research. Here, we on the first effort introduced the uracil bases into the rolling circle amplification (RCA) reaction to produce the functionalized pure DNA hydrogel (PDH) for UDG detection. During RCA process, methylene blue (MB) molecules as the indicators were encapsulated into PDH. The addition of UDG can remove the uracil bases of PDH to generate abasic sites, which are further cleaved with the assistance of apurinic/apyrimidinic endonuclease (APE), thus resulting in the dissociation of PDH to release blue MB. By combining with the paper analytical devices as the signal readout platform, a colorimetric and electrochemical dual-signal biosensor was constructed for convenient and accurate detection of UDG. The proposed MB@PDH-based dual-signal sensing system exhibited good selectivity and high sensitivity with a detection limit of 6.4 × 10−4 U/mL (electrochemical method). It was also demonstrated that this sensing system showed excellent performance in UDG inhibitor screening, thus providing great potential in UDG-related disease diagnosis and drug discovery.

A Multimodal Approach towards Genomic Identification of Protein Inhibitors of Uracil-DNA Glycosylase.

DNA-mimicking proteins encoded by viruses can modulate processes such as innate cellular immunity. An example is Ung-family uracil-DNA glycosylase inhibition, which prevents Ung-mediated degradation via the stoichiometric protein blockade of the Ung DNA-binding cleft. This is significant where uracil-DNA is a key determinant in the replication and distribution of virus genomes. Unrelated protein folds support a common physicochemical spatial strategy for Ung inhibition, characterised by pronounced sequence plasticity within the diverse fold families. That, and the fact that relatively few template sequences are biochemically verified to encode Ung inhibitor proteins, presents a barrier to the straightforward identification of Ung inhibitors in genomic sequences. In this study, distant homologs of known Ung inhibitors were characterised via structural biology and structure prediction methods. A recombinant cellular survival assay and in vitro biochemical assay were used to screen distant variants and mutants to further explore tolerated sequence plasticity in motifs supporting Ung inhibition. The resulting validated sequence repertoire defines an expanded set of heuristic sequence and biophysical signatures shared by known Ung inhibitor proteins. A computational search of genome database sequences and the results of recombinant tests of selected output sequences obtained are presented here.

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.

An ultra-sensitive and specific UCBiosensor via CRISPR-Cas12a and UDG-mediated polymerase chain reaction

DNA damage is an important cause of several diseases. Uracil DNA glycosylase (UDG) is a key enzyme in DNA damage repair and used as an early diagnostic marker and a prognostic indicator for cancer. Therefore, there is an urgent need to develop a simple, low-background, ultra-sensitive UDG activity detection method for clinical applications. At present, biosensors based on CRISPR-Cas12a have become a powerful diagnostic tool owing to their high sensitivity. Here, we have established an ultra-sensitive biosensor for detecting UDG activity based on CRISPR-Cas12a coupled UDG-mediated polymerase chain reaction (U-PCR), which has the advantages of simplicity, low background, ultra-sensitivity, and specificity. We used this biosensor to multiply amplify the UDG signal, and the detection limit reached 3.5 × 10−6 U/mL, which is one of the most sensitive methods for UDG activity detection. Finally, we detected UDG activity in the lysates of cancer cells, indicating that this method could be used to detect UDG activity in real samples. The UDG inhibition test showed that the method we established is a useful tool for screening UDG inhibitors. We believe that the UDG activity detection biosensor established in this study has potential applications in biomedical research and clinical diagnosis.

Ultra-sensitive biosensor based on CRISPR-Cas12a and Endo IV coupled DNA hybridization reaction for uracil DNA glycosylase detection and intracellular imaging

As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a “double-edged sword” for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10−6 U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a.

Cytosine base editing in cyanobacteria by repressing archaic Type IV uracil-DNA glycosylase.

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4 to C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs.

Construction of an Entropy-Driven Dumbbell-Type DNAzyme Assembly Circuit for Lighting Up Uracil-DNA Glycosylase in Living Cells.

Sensitive monitoring of intracellular uracil-DNA glycosylase (UDG) in living cells is essential to understanding the DNA repair pathways and discovery of anticancer drugs. Herein, we demonstrate the construction of an entropy-driven dumbbell-type DNAzyme assembly circuit for lighting up UDG in living cells via the integration of entropy-driven DNA catalysis (EDC) with the DNAzyme biocatalyst. Target UDG excises the damaged uracil base, causing the breakage of detection probe and the release of trigger. The released trigger can initiate the downstream EDC reaction to form two catalytically active DNAzyme units. The resultant dual Mg2+-DNAzyme units serve as the signal transducers to cyclically cleave the fluorophore/quenched-modified reporters, generating an enhanced fluorescence signal. In contrast to the single-layered EDC method with a linear amplification, the proposed doublet EDC-DNAzyme strategy exhibits high signal gain and achieves a detection limit of 8.71 × 10-6 U/mL. Notably, this assay can be performed in one-step manner at room temperature without the requirement of strict temperature control and complicated reaction procedures, and it can further screen the UDG inhibitors, measure kinetic parameters, and discriminate cancer cells from normal cells. Moreover, this strategy can monitor intracellular UDG activity with improved signal gain, and it may be exploited for sensing and imaging of other types of DNA modifying enzymes with the integration of the corresponding detection substrate, providing a facile and robust approach for biological research studies and clinical diagnosis.

Identification of a new and diverse set of Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng) inhibitors using structure-based virtual screening: Experimental validation and molecular dynamics studies

Mycobacterium tuberculosis uracil-DNA glycosylase (MtUng), a key DNA repair enzyme, represents an attractive target for the design of new antimycobacterial agents. However, only a limited number of weak MtUng inhibitors are reported, primarily based on the uracil ring, and hence, lack diversity. We report the first structure-based virtual screening (SBVS) using three separate libraries consisting of uracil and non-uracil small molecules, together with the FDA-approved drugs. Twenty diverse virtual hits with the highest predicted binding were procured and screened using a fluorescence-based assay to evaluate their potential to inhibit MtUng. Several of these molecules were found to inhibit MtUng activity at low mM and µM levels, comparable to or better than several other reported Ung inhibitors. Thus, these molecules represent a diverse set of scaffolds for developing next-generation MtUng inhibitors. The most active uracil-based compound 5 (IC50 = 0.14 mM) was found to be ∼ 15-fold more potent than the positive control, uracil. The binding stability and conformation of compound 5 in complex with the enzyme were further confirmed using molecular dynamics simulation.

PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G Base Editors for Programmable Base Editing in Mycobacteria

New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable, and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis ( M. tuberculosis ). Clustered regularly interspaced short palindromic repeat (CRISPR) base editors allow for rapid, robust, and programmed single-base substitutions and gene inactivation, yet no such systems are currently available in M. tuberculosis . By screening distinct CRISPR base editors, we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9 (St1Cas9) cytosine base editor (CBE)—but not the widely used Streptococcus pyogenes Cas9 (SpCas9) or Lachnospiraceae bacterium Cpf1 (LbCpf1) CBEs—is active in mycobacteria. Despite the notable C-to-T conversions, a high proportion of undesired byproducts exists with St1Cas9 CBE. We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor (UGI) or uracil DNA glycosylase (UNG) fusion, yielding two new base editors (CTBE and CGBE) capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis ( M. smegmatis ). Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif (PAM) sequence for DNA targeting, we engineered a PAM-expanded St1Cas9 variant by means of structure-guided protein engineering for the base editors, substantially broadening the targeting scope. We first developed and characterized CTBE and CGBE in M. smegmatis , and then applied CTBE for genome editing in M. tuberculosis . Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics, antibiotic-resistant mechanism study, and drug-target exploration in M. tuberculosis and related organisms.

HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing

Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.

Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis.

Summary Pseudomonas chlororaphis is a non‐pathogenic, plant growth‐promoting rhizobacterium that secretes phenazine compounds with broad‐spectrum antibiotic activity. Currently available genome‐editing methods for P. chlororaphis are based on homologous recombination (HR)‐dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ‐Red–like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time‐consuming, laborious and inefficient. Here, we established a CRISPR‐assisted base‐editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced‐specificity Cas9 nickase (eSpCas9ppD10A) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO‐inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR‐based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non‐traditional bacterial hosts.

Activities of endogenous APOBEC3s and uracil-DNA-glycosylase affect the hypermutation frequency of hepatitis B virus cccDNA.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) plays a key role in the persistence of viral infection. We have previously shown that overexpression of an antiviral factor APOBEC3G (A3G) induces hypermutation in duck HBV (DHBV) cccDNA, whereas uracil-DNA-glycosylase (UNG) reduces these mutations. In this study, using cell-culture systems, we examined whether endogenous A3s and UNG affect HBV cccDNA mutation frequency. IFNγ stimulation induced a significant increase in endogenous A3G expression and cccDNA hypermutation. UNG inhibition enhanced the IFNγ-mediated hypermutation frequency. Transfection of reconstructed cccDNA revealed that this enhanced hypermutation caused a reduction in viral replication. These results suggest that the balance of endogenous A3s and UNG activities affects HBV cccDNA mutation and replication competency.

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.