Rapid and simultaneous detection of common childhood diarrhea viruses by microfluidic-FEN1-assisted isothermal amplification with ultra-high specificity and sensitivity.

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea-rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/µL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis.

Synergistic powering of DNA walker movement by endogenous dual enzymes for constructing dual-mode biosensors.

To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01-500 U mL-1, presenting a good linearity in the concentration range of 0.01-10 U mL-1, with a detection limit of 0.01 U mL-1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01-50 U mL-1 and reducing the detection limit to 0.002 U mL-1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.

Gallic acid-loaded chitosan nanoparticles enhance the DNA damage and apoptotic features through inhibiting flap endonuclease-1 in triple-negative breast cancer cells.

This study investigated the fabrication of gallic acid-loaded chitosan nanoparticles (Gal-Chi-NPs) that enhanced the DNA damage and apoptotic features by inhibiting FEN-1 expressions in MDA-MB 231 cells. Gal-Chi-NPs were fabricated by the ionic gelation method, and it was characterized by several studies such as dynamic light spectroscopy, Fourier-transforms infrared spectroscopy, x-ray diffraction, scanning electron microscopy, energy-dispersive x-ray, atomic force microscopy, and thermogravimetric analysis. We have obtained that Gal-Chi-NPs displayed 182.2 nm with crystal, smooth surface, and heat stability in nature. Gal-Chi-NPs induce significant toxicity in MDA-MB-231 cells that compared with normal NIH-3T3 cells. A significant reactive oxygen species (ROS) overproduction was observed in Gal-Chi-NPs treated MDA-MB-231. Flap endonuclease-1 (FEN-1) is a crucial protein involved in long patch base excision repair that is involved in repairing the chemotherapeutic mediated DNA-damaged base. Therefore, inhibition of FEN-1 protein expression is a crucial target for enhancing chemotherapeutical efficacy. In this study, we have obtained that Gal-Chi-NPs treatment enhanced the DNA damage by observing increased p-H2AX, PARP1; and suppressed the expression of FEN-1 in MDA-MB-231 cells. Moreover, Gal-Chi-NPs inhibited the expression of tumor proliferating markers p-PI3K, AKT, cyclin-D1, PCNA, and BCL-2; induced proapoptotic proteins (Bax and caspase-3) in MDA-MB 231 cells. Thus, Gal-Chi-NPs induce DNA damage and apoptotic features and inhibit tumor proliferation by suppressing FEN-1 expression in triple-negative breast cancer cells.

Target mediated bioreaction to engineer surface vacancy effect on Bi2O2S nanosheets for photoelectrochemical detection of FEN1.

Photoelectrochemistry represents a promising technique for bioanalysis, though its application for the detection of Flap endonuclease 1 (FEN1) has not been tapped. Herein, this work reports the exploration of creating oxygen vacancies (Ov) in situ onto the surface of Bi2O2S nanosheets via the attachment of dopamine (DA), which underlies a new anodic PEC sensing strategy for FEN1 detection in label-free, immobilization-free and high-throughput modes. In connection to the target-mediated rolling circle amplification (RCA) reaction for modulating the release of the DA aptamer to capture DA, the detection system showed good performance toward FEN1 analysis with a linear detection range of 0.001-10 U/mL and a detection limit of 1.4 × 10-4 U/mL (S/N = 3). This work features the bioreaction engineered surface vacancy effect of Bi2O2S nanosheets as a PEC sensing strategy, which allows a simple, easy to perform, sensitive and selective method for the detection of FEN1. This sensing strategy might have wide applications in versatile bioasssays, considering the diversity of a variety of biological reactions may produce the DA aptamer.

Construction of single-molecule counting-based biosensors for DNA-modifying enzymes: A review.

DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 - 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors.

FEN1 upregulation mediated by SUMO2 via antagonizing proteasomal degradation promotes hepatocellular carcinoma stemness.

PURPOSE: Metastasis of hepatocellular carcinoma (HCC) critically impacts the survival prognosis of patients, with the pivotal role of hepatocellular carcinoma stem cells in initiating invasive metastatic behaviors. The Flap Endonuclease 1 (FEN1) is delineated as a metallonuclease, quintessential for myriad cellular processes including DNA replication, DNA synthesis, DNA damage rectification, Okazaki fragment maturation, baseexcision repair, and the preservation of genomic stability. Furthermore, it has been recognized as an oncogene in a diverse range of malignancies. Our antecedent research has highlighted a pronounced overexpression of protein FEN1 in hepatocellular carcinoma, where it amplifies the invasiveness and metastatic potential of liver cancer cells. However, its precise role in liver cancer stem cells (LCSCs) remains an enigma and requires further investigation. METHODS: To rigorously evaluate the stemness attributes of LCSCs, we employed sphere formation assays and flow cytometric evaluations. Both CD133+ and CD133- cell populations were discerningly isolated utilizing immunomagnetic bead separation techniques. The expression levels of pertinent genes were assayed via real-time quantitative PCR (RT-qPCR) and western blot analyses, while the expression profiles in hepatocellular carcinoma tissues were gauged using immunohistochemistry. Subsequent immunoprecipitation, in conjunction with mass spectrometry, ascertained the concurrent binding of proteins FEN1 and Small ubiquitin-related modifier 2 (SUMO2) in HCC cells. Lastly, the impact of SUMO2 on proteasomal degradation pathway of FEN1 was validated by supplementing MG132. RESULTS: Our empirical findings substantiate that protein FEN1 is profusely expressed in spheroids and CD133+ cells. In vitro investigations demonstrate that the upregulation of protein FEN1 unequivocally augments the stemness of LCSCs. In a congruent in vivo context, elevation of FEN1 noticeably enhances the tumorigenic potential of LCSCs. Conversely, inhibiting protein FEN1 resulted in a marked reduction in LCSC stemness. From a mechanistic perspective, there exists a salient positive correlation between the protein expression of FEN1 and SUMO2 in liver cancer tissues. Furthermore, the level of SUMO2-mediated modification of FEN1 is pronouncedly elevated in LCSCs. Interestingly, SUMO2 has the ability to bind to FEN1, leading to a inhibition in the proteasomal degradation pathway of FEN1 and an enhancement in its protein expression. However, it is noteworthy that this interaction does not affect the mRNA level of FEN1. CONCLUSION: In summation, our research elucidates that protein FEN1 is an effector in augmenting the stemness of LCSCs. Consequently, strategic attenuation of protein FEN1 might proffer a pioneering approach for the efficacious elimination of LCSCs.

FEN1 Promotes Hepatocellular Carcinoma Progression by Activating Cell Cycle Transition from G2 To M Phase.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that is involved in the occurrence and development of various types of tumors. Previous studies have shown that FEN1 plays an important role in the development of hepatocellular carcinoma, however, the molecular mechanisms remain fully elucidated, especially its effect on the cell cycle of hepatocellular carcinoma has not been investigated. In this study, via bioinformatics prediction and clinical specimen verification, we confirmed that FEN1 was highly expressed in HCC and correlated with poor prognosis. The knockdown or overexpression of FEN1 could inhibit or promote the proliferation and invasion of HCC cells. Importantly, cell cycle and functional experiments showed that FEN1 could promote cell proliferation by inducing cell cycle transition from G2 to M phase. Further studies indicated that FEN1 regulated the G2/M transition by modulating cell division cycle 25C (Cdc25C), cyclin-dependent kinase 1 (CDK1) and Cyclin B1 expressions. To sum up, our research suggested that FEN1 could promote the proliferation, migration and invasion of HCC cells via activating cell cycle progression from G2 to M phase, indicating that FEN1 may be a potential target for the treatment of HCC.

A FEN 1-driven DNA walker-like reaction coupling with magnetic bead-based separation for specific SNP detection.

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5'flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.

A one-pot transcriptional assay method that detects the tumor biomarker FEN1 based on its flap cleavage activity.

BACKGROUND: Detection of tumor biomarkers in body fluids is a significant advancement in cancer treatment because it allows diagnosis without invasive tissue biopsies. Nucleases have long been regarded as a potential class of biomarkers that can indicate the occurrence and progression of cancers. Among these, flap endonuclease 1 (FEN1) plays an important role in DNA replication and repair, and also overexpressed in abnormally proliferating cells such as cancer cells. FEN1 is thus considered to be a potential biomarker as well as a target for cancer therapy. RESULTS: We developed a novel method for detecting FEN1 based on its specific endonuclease activity which incises bifurcated nucleic acids (flaps), in combination with in vitro transcription. Developed method uses a simple DNA structure (substrate DNA) carrying a short 5'-flap sequence, and a single-stranded sensor DNA encoding the Broccoli light-up aptamer. When the assay mixture was supplied with a FEN1-containing sample, the flap sequence encoding the sense sequence of T7 promoter was cleaved and released from the substrate DNA. Because the sensor DNA was designed to carry the Broccoli RNA aptamer under the antisense sequence of T7 promoter, hybridization of the excised flap onto the sensor DNA initiated the transcription of the Broccoli RNA aptamer, enabling determination of the FEN1 titer based on the fluorescence of transcribed Broccoli aptamer. By using a combination of FEN1-mediated generation of a short oligonucleotide and subsequent oligonucleotide-dependent in vitro transcription, this method could detect FEN1 in biological samples within 1 h. SIGNIFICANCE AND NOVELTY: Developed method enables the detection of FEN1 by a simple one-pot reaction. It can detect sub-nanomolar concentrations of FEN1 within an hour, and has the potential to be used for cancer diagnosis, prognosis, and drug screening. It also enables easy identification of compounds that inhibit FEN1 activity and is thus a versatile platform for screening anti-cancer drugs. We anticipate that the basic principles of this assay can be applied to detect other biomolecules, such as nucleic acids.

Combination of downregulating FEN1 and PD-1 blockade enhances antitumor activity of CD8+ T cells against HNSCC cells in vitro.

BACKGROUND: Programmed cell death ligand 1 (PD-L1) and human leukocyte antigen/major histocompatibility complex (HLA/MHC) are two main kinds of immunophenotypes affecting the susceptibility to anti-PD therapy. Our previous study found that down-regulation of flap endonuclease-1 (FEN1) could not only inhibit PD-L1 expression, but also upregulate HLA expression in head and neck squamous cell carcinoma (HNSCC). We aimed to clarify whether downregulating FEN1 cloud enhance the response to PD-1 blockade, and possible mechanisms in HNSCC in vitro. METHODS: Differential expression of FEN1 in HNSCC tumor and normal tissues were explored in the TIMER and TISIDB datasets. A HNSCC cells/CD8+ T cells co-culture model was established. HNSCC cell cycle and apoptosis were recorded by flow cytometry. Immune activity markers of granzyme A, granzyme B, and PRF1 expressed in the CD8+ T cells, and IFN-γ, IL-2, and TNF-α secreted in the supernatants were detected by western blot, ELISA, respectively. RESULTS: FEN1 was highly expressed in HNSCC and associated with low immune infiltration. Downregulating FEN1 could induce HLA class I expression, and inhibit PD-L1 expression in HNSCC cells. Functionally, FEN1 knockdown enhanced the response to αPD-1 mAb by mediating G2/M phase arrest, apoptosis of HNSCC cells. Mechanistically, targeting FEN1 synergized with αPD-1 mAb could reinforce the antitumor response of CD8+ T cells against HNSCC cells, as indicated by increasing granzyme A, granzyme B, and PRF1 expressions, and promoting IFN-γ, IL-2, and TNF-α secretions. CONCLUSION: These findings might offer a potential combined strategy for patients resistant to anti-PD therapy via combining FEN1 knockdown and PD-1 blockade.

Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK/ELK1 signalling pathway.

PURPOSE: Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. METHODS: Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. RESULTS: Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. CONCLUSION: Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.

Construction of dual exponential amplification accompanied by multi-terminal signal output method for convenient detection of tumor biomarker FEN1 activity.

As an important 5'-nuclease in DNA replication and damage repair, Flap endonuclease 1 (FEN1) has been considered as a potential tumor biomarker due to its overexpression in different human cancer cells. Here, we developed a convenient fluorescent method based on dual enzymatic repairing exponential amplification accompanied by multi-terminal signal output to realize the rapid and sensitive detection of FEN1. In the presence of FEN1, the double-branched substrate could be cleaved to produce 5' flap single strand DNA (ssDNA) which subsequently was used as a primer to initiate the dual exponential amplification (EXPAR) to generate abundant ssDNAs (X' and Y'), then the ssDNAs can respectively hybridize with the 3' and 5' ends of the signal probe to form partially complementary double strands (dsDNAs). Subsequently, the signal probe on the dsDNAs could be digested under the assistance of Bst. polymerase and T7 exonuclease, as well as releasing the fluorescence signals. The method displayed high sensitivity with the detection limit of 9.7 × 10-3 U mL-1 (1.94 × 10-4 U) and also exhibited good selectivity towards FEN1 under the challenge from complicated samples including extracts of normal and cancer cells. Furthermore, it was successfully applied to screen FEN1 inhibitors, holding great promise in the screening of potential drugs targeting FEN1. This sensitive, selective and convenient method could be used for FEN1 assay without the complicated nanomaterial synthesis/modification, showing great potential in FEN1- related prediction and diagnosis.

MiR-26a-5p Heightens Breast Cancer Cell Sensitivity to Paclitaxel via Targeting Flap Endonuclease 1.

OBJECTIVE: Flap endonuclease 1 (FEN1) has been confirmed to involve the drug resistance of multiple cancers including breast cancer. However, the effect of miRNA-mediated FEN1 on breast cancer cell resistance is still ambiguous and needs further research. METHODS: Firstly, we used GEPIA2 to predict the FEN1 expression in breast cancer. Next, we used quantitative real-time polymerase chain reaction (qRT-PCR) and western blot to evaluate the FEN1 level of cells. After parental cells or MDA-MB-231-paclitaxel (PTX) cells being transfected with or without siFEN1, the apoptosis, migration, and protein levels of FEN1, Bcl-2, and resistance-related genes were examined by flow cytometry, wound healing assay, and western blot, respectively. Then, the putative miRNA targeting FEN1 was predicted using StarBase V3.0, and further confirmed by qRT-PCR. The targeted binding of FEN1 to miR-26a-5p was detected by dual-luciferase reporter assay. After parental cells or MDA-MB-231-PTX cells being transfected with or without miR-26a-5p mimic, the apoptosis, migration, and protein levels of FEN1, Bcl-2, and resistance-related genes were tested again. RESULTS: FEN1 expression was enhanced in breast cancer and MDA-MB-231-PTX cells. The combined application of FEN1 knockdown and PTX enhanced apoptosis in MDA-MB-231-PTX cells but suppressed cell migration and expressions of FEN1, Bcl-2, and resistance-related genes. Then, we confirmed that FEN1 was targeted by miR-26a-5p. The combined application of miR-26a-5p mimic and PTX largely facilitated apoptosis in MDA-MB-231-PTX cells but restrained cell migration and expressions of FEN1, Bcl-2, and resistance-related genes. CONCLUSION: MiR-26a-5p contributes to the sensitivity of breast cancer cells to paclitaxel via restraining FEN1.

Integrative analyses of biomarkers and pathways for metformin reversing cisplatin resistance in head and neck squamous cell carcinoma cells.

OBJECTIVES: In this study, transcriptome sequencing were performed to elucidate the molecular mechanism by which metformin inhibits head and neck squamous cell carcinoma (HNSCC) cells progression and sensitizes HNSCC cells to chemotherapy. We aimed to propose a novel chemotherapeutic approach with high efficacy and few side effects and provide a new strategy for HNSCC treatment. DESIGN: The effects of metformin on the biological behaviors of HNSCC cells were validated by CCK8 cell proliferation assays, would healing assays and flow cytometric apoptosis assays. The appropriate metformin concentrations for the experimental pretreatment of HNSCC cells were selected based on experimental results, and the treated cells were subjected to transcriptome sequencing. After bioinformatics analysis and intersection with a post-chemotherapy resistance dataset from the GEO database numbered GSE102787, the genes were identified and used to predict potential metformin targets after functional enrichment analysis. RESULTS: Metformin significantly inhibited the proliferation and migration and induced the apoptosis of Cal27 and FaDu cells. A total of 284 genes that are potentially targeted by metformin during HNSCC cell sensitization were identified by bioinformatics, and ten hub genes with high connectivity were selected. In particular, Fen1 overexpression was associated with poor prognosis in HNSCC patients. CONCLUSIONS: Our study demonstrates that Fen1 is overexpressed in HNSCC tissues compared with normal tissues and that Fen1 overexpression is a poor prognostic factor in HNSCC patients. Metformin enhances the ability of cisplatin to inhibit HNSCC progression. Further studies are needed to explore the therapeutic value of Fen1 in HNSCC.

Double-wing switch nanodevice-mediated primer exchange reaction for the activity analysis of cancer biomarker FEN1.

DNA damage repair is one of the foremost factors leading to changes in tumor drug resistance. The analysis of Flap endonuclease 1 (FEN1), a kind of pivotal enzyme in various DNA metabolic pathways, has been of great support to tumor research and the development of chemotherapeutics. Nevertheless, few analytical techniques can achieve quantitative and simplified FEN1 measurement. Here, we constructed a double-wing switch nanodevice (DWSN)-mediated primer exchange technique for rapid and label-free quantification of FEN1 activity. Target FEN1 triggered the generation of numerous telomeric repeat fragments in different lengths through recognizing the three-base mismatched sites on the DWSN to release the 5'-Flaps. Further binding to the fluorescent dye ThT resulted in significantly enhanced fluorescence. This study broke the limitation of traditional single-site identification and demonstrated good sensitivity and specificity with detection limits up to 0.55 mU. Besides, the extraordinary analytical performance allowed the method to be utilized to monitor FEN1 extracted from cells and clinical serum samples and to compare the effect of targeted FEN1 inhibitors.

Multimodal detection of flap endonuclease 1 activity through CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides.

Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.

Invader assay-induced catalytic assembly of multi-DNAzyme junctions for sensitive detection of single nucleotide polymorphisms.

Single nucleotide polymorphisms (SNPs) are main causes of differences in human phenotypes and drug resistance, especially the development of genetic diseases. Monitoring SNPs is of significant benefit to drug design and disease diagnosis. In this work, on the basis of tripartite DNAzyme junction formation induced by flap endonuclease 1 (FEN1) invader assay-triggered catalytic hairpin assembly (CHA), we describe an amplified and highly sensitive fluorescent strategy for detecting SNP of K-ras gene with substantial discrimination capability. The mutant DNA (MtDNA) of K-ras gene hybridizes with the sensing probe to inhibit the enzymatic activity of FEN1 to trigger subsequent CHA of three hairpins for the formation of tripartite DNAzyme junctions. And, the fluorescently quenched signal probes are efficiently and cyclically cleaved by the DNAzymes to restore largely magnified fluorescence for detecting MtDNA target sequence at 4.23 fM. Besides, the existence of low levels of MtDNA strands in diluted human serums and high concentrations of wild DNA solutions can be differentiated by such a method, showing its high potential for monitoring various SNPs for biological research and disease diagnosis.

Conversion of hepatitis B virus relaxed circular to covalently closed circular DNA is supported in murine cells.

Background & Aims: HBV has a narrow host restriction, with humans and chimpanzees representing the only known natural hosts. The molecular correlates of resistance in species that are commonly used in biomedical research, such as mice, are currently incompletely understood. Expression of human NTCP (hNTCP) in mouse hepatocytes enables HBV entry, but subsequently covalently closed circular (cccDNA) does not form in most murine cells. It is unknown if this blockade in cccDNA formation is due to deficiency in repair of relaxed circular DNA (rcDNA) to cccDNA. Methods: Here, we deployed both in vivo and in vitro virological and biochemical approaches to investigate if murine cells contain a complete set of repair factors capable of converting HBV rcDNA to cccDNA. Results: We demonstrate that HBV cccDNA does form in murine cell culture or in mice when recombinant rcDNA without a protein adduct is directly introduced into cells. We further show that the murine orthologues of core components in DNA lagging strand synthesis, required for the repair of rcDNA to cccDNA in human cells, can support this crucial step in the HBV life cycle. It is worth noting that recombinant HBV rcDNA substrates, either without a protein adduct or containing neutravidin to mimic HBV polymerase, were used in our study; it remains unclear if the HBV polymerase removal processes are the same in mouse and human cells. Conclusions: Collectively, our data suggest that the HBV life cycle is blocked post entry and likely before the repair stage in mouse cells, which yields critical insights that will aid in the construction of a mouse model with inbred susceptibility to HBV infection. Lay summary: Hepatitis B virus (HBV) is only known to infect humans and chimpanzees in nature. Mouse models are often used in modeling disease pathogenesis and preclinical research to assess the efficacy and safety of interventions before they are then tested in human participants. However, because mice are not susceptible to HBV infection it is difficult to accurately model human infection (and test potential treatments) in mouse models. Herein, we have shown that mice are able to perform a key step in the HBV life cycle, tightening the net around the possible reason why HBV can not efficiently infect and replicate in mice.

Label-Free and Homogeneous Electrochemical Biosensor for Flap Endonuclease 1 Based on the Target-Triggered Difference in Electrostatic Interaction between Molecular Indicators and Electrode Surface.

Target-induced differences in the electrostatic interactions between methylene blue (MB) and indium tin oxide (ITO) electrode surface was firstly employed to develop a homogeneous electrochemical biosensor for flap endonuclease 1 (FEN1) detection. In the absence of FEN1, the positively charged methylene blue (MB) is free in the solution and can diffuse onto the negatively charged ITO electrode surface easily, resulting in an obvious electrochemical signal. Conversely, with the presence of FEN1, a 5'-flap is cleaved from the well-designed flapped dumbbell DNA probe (FDP). The remained DNA fragment forms a closed dumbbell DNA probe to trigger hyperbranched rolling circle amplification (HRCA) reaction, generating plentiful dsDNA sequences. A large amount of MB could be inserted into the produced dsDNA sequences to form MB-dsDNA complexes, which contain a large number of negative charges. Due to the strong electrostatic repulsion between MB-dsDNA complexes and the ITO electrode surface, a significant signal drop occurs. The signal change (ΔCurrent) shows a linear relationship with the logarithm of FEN1 concentration from 0.04 to 80.0 U/L with a low detection limit of 0.003 U/L (S/N = 3). This study provides a label-free and homogeneous electrochemical platform for evaluating FEN1 activity.

Fluorescence imaging of FEN1 activity in living cells based on controlled-release of fluorescence probe from mesoporous silica nanoparticles.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease, which catalyzes the removal of 5' overhanging DNA flap from a specific DNA structure. FEN1 has been considered as an important biomarker for cancer diagnosis since it is over-expressed in various types of human tumor cells and closely related to cancer development. Nanoprobes gradually become basic tools for analyzing biomarkers variations in vivo. Here, we utilized aminoated mesoporous silica nanoparticles (NH2-MSNs) with a rich porous structure as the fluorescence nanoprobes to entrap the rhodamine 6G (Rh6G) molecules. Then gold nanoparticles linked specific single-stranded DNA (AuNPs-ssDNA) as a molecular gate was used to coat the NH2-MSNs surface. The fluorescence signal was weak when the fluorescence molecules were blocked by the AuNPs-ssDNA. In the presence of FEN1, it recognized and cleaved the specific ssDNA to release the Rh6G from NH2-MSNs, which resulted in recovered fluorescence signals. Thus, the sensitive detection of FEN1 activity was realized by controlled-release of Rh6G. The fluorescence signal showed a good linear relationship with the logarithm of FEN1 activity ranging from 0.05 to 1.75 U with a detection limit of 0.03 U. Moreover, confocal imaging demonstrated that the proposed biosensor could distinguish tumor cells from normal cells. Therefore, this technique contributes to clinical diagnostic and therapeutic monitoring.