Target-induced Trivalent G-quadruplex/hemin DNAzyme for Colorimetric Detection of Hg^(2+) Based on an Exonuclease III Assisted Catalytic Hairpin Assembly

Mercury ion(Hg^(2+)),a highly noxious of heavy metalion,has detrimental effects on the ecological environment and human health.Herein,we have developed an exonuclease III(Exo III)assisted catalytic hairpin assembly formation of a trivalent G-quadruplex/hemin DNAzyme for colorimetric detection of Hg^(2+).A hairpin DNA(Hr)was designed with thymine-Hg^(2+)-thymine pairs that catalyzed by Exo III is prompted to happen upon binding Hg^(2+).A released DNA fragment triggers the catalytic assembly of other three hairpins(H1,H2,and H3)to form many trivalent G-quadruplex/hemin DNA enzymes for signal output.The developed sensor shows a dynamic range from 2 pM to 2μM,with an impressively low detection limit of 0.32 pM for Hg^(2+)detection.Such a sensor also has good selectivity toward Hg^(2+)detection in the presence of other common metal ions.This strategy shows the great potential for visual detection with portable type.

Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD:AhR:DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

Triple signal amplification electrochemical sensing platform for Hg^(2+) in water without direct modification of the working electrode

An ultrasensitive electrochemical biosensor to detect trace Hg^(2+)in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide(MrGO-AuNPs),exonuclease III-assisted target cycle(Exo Ⅲ-ATC)and hybridization chain reaction(HCR)synergistic triple signal amplification.The MrGO-AuNPs is a superior carrier for capture DNA(cDNA)and acts as magnetic media for automatic separation and adsorption.This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode.Additionally,the three DNA hairpins(cDNA,methylene blue(MB)labeled HP1 and HP2)further contribute to biosensor specificity and selectivity.When cDNA captures Hgt,it activates Exo Ⅲ-ATC due to the formation of a sticky end in the DNA stem via thymine-Hig-thymidine(T-Hg^(2+)-T),this leads to the hydrolysis of self-folded DNA by Exo Ⅲ-ATC to form"key"DNA(kDNA).The kDNA subsequently initiates HCR,resulting in massive super-sandwich structures(kDNA-[HP1/HP2])carrying signaling molecules on MrGO-AuNPs,and this overall structure serves as a signal probe(SP).Leveraging magnetic adsorption,the SP was automatically adsorbed onto the magneto-glass carbon electrode(MGCE),generating an amplified signal.This biosensor's detection limit(LOD)was 3.14 pmol/L,far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA.The biosensor also showed excellent selectivity when challenged by interfering ions,and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring.

Half a century after their discovery:Structural insights into exonuclease and annealase proteins catalyzing recombineering

Recombineering is an essential tool for molecular biologists,allowing for the facile and efficient manipulation of bacterial genomes directly in cells without the need for costly and laborious in vitro manipulations involving restriction enzymes.The main workhorses behind recombineering are bacteriophage proteins that promote the single-strand annealing(SSA)homologous recombination pathway to repair double-stranded DNA breaks.While there have been several reviews examining recombineering methods and applications,comparatively few have focused on the mechanisms of the proteins that are the key players in the SSA pathway:a 5′→3′exonuclease and a single-strand annealing protein(SSAP or“annealase”).This review dives into the structures and functions of the two SSA recombination systems that were the first to be developed for recombineering in E.coli:the RecET system from E.coli Rac prophage and the𝜆Red system from bacteriophageλ.By comparing the structures of the RecT and Red𝛽annealases,and the RecE and𝜆Exo exonucleases,we provide new insights into how the structures of these proteins dictate their function.Examining the sequence conservation of the𝜆λExo and RecE exonucleases gives more profound insights into their critical functional features.Ultimately,as recombineering accelerates and evolves in the laboratory,a better understanding of the mechanisms of the proteins behind this powerful technique will drive the development of improved and expanded capabilities in the future.

Addition of the T5 exonuclease increases the prime editing efficiency in plants

Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.

A novel loss-of-function variant in PNLDC1 inducing oligo-astheno-teratozoospermia and male infertility

Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.

Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites

CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease(T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.

My Journey to DNA Repair

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have dis- covered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases I, III and IV. I discovered the mam- malian exonucleases DNase III （TREX1） and IV （FEN1）. Interestingly, expression of TREXI was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine （O6mG） is repaired without removing the guanine from DNA, identifying a sur- prising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.

An AIEgens and exonuclease Ⅲ aided quadratic amplification assay for detecting and cellular imaging of telomerase activity

Monitoring telomerase activity with high sensitive and reliable is of great importance to cancer analysis. In this paper, we report a sensitive and facile method to detect telomerase activity using AIEgens mod- ified probe （TPE-Py-DNA） as a fluorescence reporter and exonuclease llI （Exo lIl） as a signal amplifier. With the aid of telomerase, repeat units （TrAGGG）n are extended from the end of template substrate oligonucleotides （TS primer） that form duplex DNAs with TPE-Py-DNA. Then, Exo llI catalyzes the diges- tion of duplex DNAs, liberating elongation product and releasing hydrophobic TPE-Py. The released hydrophobic TPE-Py aggregate together and produce a telomerase-activity-related fluorescence signal. The liberated product hybridizes with another TPE-Py-DNA probe, starting the second cycle. Finally, we obtain the target-to-signal amplification ratio of 1 ：N2. This strategy exhibits good performance for detecting clinical urine samples （distinguishing 15 cancer patients＇ samples from 8 healthy ones） and checking intracellular telomerase activity （differentiating cell lines including HeLa, MDA-MB-231, MCF-7, A375, HLF and MRC-5 from the cells pretreated with telomerase-related drug）, which shows its potential in clinical diagnosis as well as therapeutic monitoring of cancer.

A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodyna mics aspects.The optimization of the assista nt strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90%.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1(rs1799949,c.2082 C>T).The limit of detection to the mutation abundance in the DNA three-way junction structure system(0.2%) was one order lower compared with that in the double-stranded DNA structure system(2%).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.

An ultrasensitive aptasensor for prostate specific antigen assay based on Exonuclease T-aided cyclic cleavage

An ultrasensitive electrochemical aptasensor is presented for prostate specific antigen(PSA) detection. DNA tetrahedronaptamer is designed, which not only facilitates the molecular self-assembly events,but also improves the recognition efficiency between PSA and aptamer sequence on the electrode interface. The DNA conformation on top of DNA tetrahedron changes accordingly, which can be further digested by Exonuclease T(Exo T), a type of single-strand specific nuclease. Electrochemical species are removed synchronously and the initial PSA level can thus be determined. A linear range from 0.5 pg mL^(-1) to50 ng mL^(-1) is achieved with the limit of detection(LOD) as low as 0.15 pg mL^(-1). Moreover, this proposed method is highly selective and is successfully applied to determine PSA in human serum samples.

Ultrasensitive Label-free Detection of miRNA with Asymmetric Hairpin Probe, Exonuclease I and SYBR Green I

Detection of miRNAs presents a particular challenge because of their limited size, high sequence homo- logy and greatly various expression level. In this work, an ultrasensitive, label-free and isothermal miRNA detection was developed based on asymmertric hairpin probe, exonuclease I（Exo I） and SYBR Green I. The method employed asymmetric hairpin probe to perform cycled polymerization and Exo I to reduce background signal. In the presence of the target miRNA, the target triggers probe-mediated cycled polymerization reactions to generate lots of dsDNA products. The dsDNA product effectively prevents itself from being degraded by Exo I and permitted the insertion of more fluorescence dye into it to enlarge the fluorescence signal. In the absence of the target miRNA, there was no probe-mediated polymerization reaction, and the probe was digested by Exo I added, which minimized the intercala- tion of fluorescence dye to reduce the background signal. The combination of the probe-mediated cycled polymeriza- tion with the Exo 1-assisted background reduction allows us to achieve a detection limit of 5× 10^-18 mol/L. Owing to its ultrasensitivity, excellent specificity, convenience and low-cost, this method might hold out great promise in miRNA detection.

A novel aptasensor strategy for protein detection based on G-quadruplex and exonuclease Ⅲ-aided recycling amplification

The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.

Multi-functions of exonuclease 1 in DNA damage response and cancer susceptibility

Exonuclease 1(EXO1)can catalyze nucleotide chain excision with its conserved N-terminal domain of 5′ to 3′ exonuclease activity,enabling it to influence diverse biological processes facing the challenges of genotoxic environmental factors such as ionizing radiation.This nuclease activity enables EXO1 to maintain replication forks and telomeres length,to facilitate post-replication DNA repair and to process the end resection step of homologous recombination of DNA double-strand breaks-induced by ionizing radiation.When DNA replication is disrupted or blocked,EXO1 can cleave the broken DNA ends to form 3’ssDNA,leading to repair pathways activation.Excess EXO1-mediated nucleotide excision,however,can introduce an abundance of single-stranded DNA that can cause mutation and recombination via micro-homology-mediated end joining or single-strand annealing mechanisms,contributing to a loss of genetic information.EXO1 activity must therefore be carefully regulated within healthy cells.The mutations and dysregulations of EXO1 can increase the sensitivity of cells to radiation injury and risk of oncogenic transformation,limit the adoption of specific treatments in a range of human diseases.As such,EXO1 represents a promising target for the treatment and prevention of cancer.In the present review,we delineate the structural properties and functional characteristics of EXO1,discuss the relationship between this exonuclease and cancer susceptibility as well as the second cancers related to radiotherapy.