Enzyme-powered, label-free DNA walker for Uracil-DNA glycosylase detection at single-cell level.

Uracil-DNA glycosylase (UDG) plays a crucial role in the removal of damaged uracil bases, thereby upholding genetic stability and integrity. An enzyme-powered, label-free DNA walker was devised for UDG activity detection. Initially, a label-free DNA track, incorporating a gold nanoparticle (AuNP), multiple hairpin structures, and various swing arms, was engineered for walking mechanism. The hairpin structure was meticulously crafted to include a G-quadruplex sequence, enabling the generation of a label-free fluorescence signal. The swing arm remained inert in the absence of UDG, but became activated upon the introduction of UDG, thereby initiating the enzyme-powered walking process and generating significant dissociative G-quadruplex sequences. By integrating a selective fluorescent dye into the design, an enhanced label-free fluorescence response was achieved. The proposed DNA walker presented a direct and label-free approach for UDG detection, demonstrating exceptional sensitivity with a detection limit of 0.00004 U/mL. Using the uracil glycosylase inhibitor (UGI) as an inhibitory model, inhibitor assay was conducted with satisfactory precision. Furthermore, successful analysis of cellular UDG at the single-cell level was accomplished. Consequently, the developed DNA walker serves as a label-free, selective, and sensitive tool for UDG activity assessment, showing great potential for applications in disease diagnosis, inhibitor screening, and biomedical investigations.

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

BACKGROUND: As 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. RESULTS: We 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. SIGNIFICANCE: URECA 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.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination.

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

Distortion of Population Statistics due to the Use of Different Methodological Approaches to the Construction of Genomic DNA Libraries.

Several different methods of DNA library preparation for paleogenetic studies are now available. However, the chemical reactions underlying each of them can affect the primary sequence of ancient DNA (aDNA) in the libraries and taint the results of a statistical analysis. In this paper, we compare the results of a sequencing of the aDNA libraries of a Bronze Age sample from burials of the Caucasian burial ground Klady, prepared using three different approaches: (1) shotgun sequencing, (2) strategies for selecting target genomic regions, and (3) strategies for selecting target genomic regions, including DNA pre-treatment with a mixture of uracil-DNA glycosylase (UDG) and endonuclease VIII. The impact of the studied approaches to genomic library preparation on the results of a secondary analysis of the statistical data, namely F4 statistics, ADMIXTURE, and principal component analysis (PCA), was analyzed. It was shown that preparation of genomic libraries without the use of UDG can result in distorted statistical data due to postmortem chemical modifications of the aDNA. This distortion can be alleviated by analyzing only the single nucleotide polymorphisms caused by transversions in the genome.

Photoelectrochemical polarity-switching-mode and split-type biosensor based on SQ-COFs/BiOBr heterostructure for the detection of uracil-DNA glycosylase.

Here, we constructed a split-type and photocurrent polarity switching photoelectrochemical (PEC) biosensor for ultrasensitive detection of Uracil-DNA glycosylase (UDG, abnormal UDG activity is correlated with human immunodeficiency, cancers, bloom syndrome, neurodegenerative diseases and so on) based on SQ-COFs/BiOBr heterostructure, as the photoactive materials, methylene blue (MB) as the signal sensitizer, and catalytic hairpin assembly (CHA) for signal amplification. Specifically, the photocurrent intensity generated by SQ-COFs/BiOBr was about 2 and 6.4 times of that of BiOBr and SQ-COFs alone, which could be responsible for the detection sensitivity for the proposed biosensor. In addition, it is not common to construct heterojunctions between covalent organic skeletons and inorganic nanomaterials. In UDG recognition tube, the plenty of COP probes loaded methylene blue (MB) were obtained by magnetic separation with the help of the simple chain displacement reaction of CHA. MB, as a responsive substance, can efficiently switched the photocurrent polarity of the SQ-COFs/BiOBr electrode from cathode to anode, which reduce the background signal, further improve the sensitivity of the biosensor. Based on the above, the linear detection range of our designed biosensor is 0.001-3 U mL-1, and the detection limit (LODs) is as low as 4.07 × 10-6 U mL-1. Furthermore, the biosensor can still maintain good analytical performance for UDG in real sample, which means that it has broad application prospects in the field of biomedicine.

Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.

Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

Generation of 3'-OH terminal-triggered encoding of multicolor fluorescence for simultaneous detection of different DNA glycosylases.

Uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG) are the important DNA glycosylases for initiating the repair of DNA damage, and the aberrant expression of DNA glycosylases is closely associated with various diseases, such as Parkinson's disease, several cancers, and human immunodeficiency. The simultaneous detection of UDG and hAAG is helpful for the study of early clinical diagnosis. However, the reported methods for multiple DNA glycosylase assay suffer from the application of an expensive single-molecule instrument, labor-tedious magnetic separation, and complicated design. Herein, we develop a simple fluorescence method with only three necessary DNA strands for the selective and sensitive detection of multiple DNA glycosylase activity based on the generation of 3'-OH terminal-triggered encoding of multicolor fluorescence. The method can achieve the detection limits of 5.5 × 10-5 U/mL for UDG and 3.3 × 10-3 U/mL for hAAG, which are lower than those of the reported fluorescence methods. Moreover, it can be further used to detect multiple DNA glycosylases in the human cervical carcinoma cell line (HeLa cells), normal human renal epithelial cells (293 T cells), and biological fluid and measure the enzyme kinetic parameters of UDG and hAAG.

Circ-Udg Derived from Cyprinid Herpesvirus 2 Promotes Viral Replication.

Cyprinid herpesvirus 2 (CyHV-2) has caused great losses to the gibel carp (Carassius auratus gibelio) industry. Previous studies showed that certain DNA viruses can encode circular RNAs (circRNAs) to regulate virus infection, which provides new clues for the treatment of viral disease. Whether CyHV-2 can encode circRNAs is still unknown. Here, 10 CyHV-2-derived circRNAs were identified, and the function of circ-udg, a circRNA derived from the CyHV-2 uracil DNA glycosylase (udg) gene, was studied. Although the expression level of circ-udg was lower than that of the parental gene, udg, its expression level was elevated in tandem with the proliferation of CyHV-2 and udg. In vitro experiments confirmed that circ-udg could promote the proliferation of CyHV-2. Moreover, circ-udg could encode a truncated UDG protein consisting of 147-amino-acid residues (termed circ-udg-P147). Both UDG and circ-udg-P147 were found to promote CyHV-2 proliferation, but the promoting effect of circ-udg on CyHV-2 proliferation was attenuated after circ-udg lost the ability to encode circ-udg-P147. Also, circ-udg-P147 could not change the transcription level of the udg gene. Interestingly, the UDG protein level was increased by circ-udg-P147. These results deepen the understanding of the genetic information carried by the genome of CyHV-2 and provide a new target for the treatment of gibel carp bleeding disease caused by CyHV-2. IMPORTANCE The outbreak of C. auratus gibelio gill hemorrhagic disease caused by CyHV-2 brought great losses to the gibel carp industry. Therefore, exploring the interaction between CyHV-2 and host and the molecular mechanism of viral infection is of great significance in preventing and treating the gibel carp gill hemorrhagic disease. Although some progress has been made in the study of CyHV-2, the mechanism of interaction between CyHV-2 and crucian carp is still unclear. In this study, we found that CyHV-2 can encode circRNA to regulate virus replication. Our study provides novel information on CyHV-2 functional genomics, a reference for research into the circRNA of other viruses, and theoretical guidance for preventing and treating gibel carp bleeding disease.

Preparation of DNA interstrand cross-link repair intermediates induced by abasic sites.

DNA interstrand cross-links (ICLs) are extremely deleterious DNA lesions, which can block different DNA transactions. A major step in ICL repair involves strand cleavage activities flanking the cross-linking site, also known as unhooking. The cleavage generates a single-stranded DNA remnant attached to the unbroken strand, often referred to as the unhooked ICL repair intermediates. The unhooked ICLs are substrates for specialized DNA polymerases, leading to the eventual restoration of the duplex DNA structure. Although these repair events have been outlined, the understanding of molecular details of the repair pathways has been hindered by the difficulty of preparing structurally defined ICL repair intermediates. Here, we present a straightforward method to prepare model ICL repair intermediates derived from a ubiquitous type of endogenous DNA modification, abasic (AP) sites. AP-derived ICLs have emerged as an important type of endogenous ICLs. We developed the method based on commercially available materials without the requirement of synthetic chemistry expertise. The method is expected to be accessible to any interested labs in the DNA repair community. • The method exploits the alkaline lability of ribonucleotides and uses designer oligonucleotides to create ICL repair intermediates with varying lengths of the unhooked strand. • Strand cleavage at ribonucleotides is achieved using NaOH, which avoids the potential for incomplete digestion during enzymatic workup due to specific substrate structures. • The method is grounded on the high cross-linking yield between an AP lesion and a nucleotide analog, 2-aminopurine, via reductive amination, developed by Gates and colleagues.

Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP).

OBJECTIVES: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix. METHODS: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method. RESULTS: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78-98.2%) and 100% specificity (95% confidence interval: 92-100%) using UDG RT-LAMP. CONCLUSION: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.

Utility of blood as the clinical specimen for the diagnosis of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification and real-time polymerase chain reaction assays based on REP-529 sequence and B1 gene.

BACKGROUND: Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1. METHODS: One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1. RESULTS: Detection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg of T. gondii genomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection of Toxoplasma gondii DNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1. CONCLUSIONS: Data from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis of ocular toxoplasmosis in blood samples of patients with active chorioretinal lesions.

Computational Study on DNA Repair: The Roles of Electrostatic Interactions Between Uracil-DNA Glycosylase (UDG) and DNA.

Uracil-DNA glycosylase (UDG) is one of the most important base excision repair (BER) enzymes involved in the repair of uracil-induced DNA lesion by removing uracil from the damaged DNA. Uracil in DNA may occur due to cytosine deamination or deoxy uridine monophosphate (dUMP) residue misincorporation during DNA synthesis. Medical evidences show that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, and liver cancer. Therefore, the research of UDG is crucial in cancer treatment and prevention as well as other clinical activities. Here we applied multiple computational methods to study UDG in several perspectives: Understanding the stability of the UDG enzyme in different pH conditions; studying the differences in charge distribution between the pocket side and non-pocket side of UDG; analyzing the field line distribution at the interfacial area between UDG and DNA; and performing electrostatic binding force analyses of the special region of UDG (pocket area) and the target DNA base (uracil) as well as investigating the charged residues on the UDG binding pocket and binding interface. Our results show that the whole UDG binding interface, and not the UDG binding pocket area alone, provides the binding attractive force to the damaged DNA at the uracil base.

An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.

Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules. We found aurintricarboxylic acid (ATA) as an inhibitor of purified UDG at an initial calculated IC50 < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC50 of 700 nM and showed direct binding to the human UDG with a KD of <700 nM. ATA displays preferential, dose-dependent binding to purified human UDG compared to human 8-oxoguanine DNA glycosylase. ATA did not bind uracil-containing DNA at these concentrations. Yet, combined crystal structure and in silico docking results unveil ATA interactions with the DNA binding channel and uracil-binding pocket in an open, destabilized UDG conformation. Biologically relevant ATA inhibition of UDG was measured in cell lysates from human DLD1 colon cancer cells and in MCF-7 breast cancer cells using a host cell reactivation assay. Collective findings provide proof-of-principle for development of an ATA-based chemotype and "door stopper" strategy targeting inhibitor binding to a destabilized, open pre-catalytic glycosylase conformation that prevents active site closing for functional DNA binding and nucleotide flipping needed to excise altered bases in DNA.

Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background.

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10-6 U/mL and 1 × 10-4 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.

Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion.

Fresh-frozen tissue is the "gold standard" biospecimen type for next-generation sequencing (NGS). However, collecting frozen tissue is usually not feasible because clinical workflows deliver formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Some clinicians and researchers are reticent to embrace the use of FFPE tissue for NGS because FFPE tissue can yield low quantities of degraded DNA, containing formalin-induced mutations. We describe the process by which formalin-induced deamination can lead to artifactual cytosine (C) to thymine (T) and guanine (G) to adenine (A) (C:G > T:A) mutation calls and perform a literature review of 17 publications that compare NGS data from patient-matched fresh-frozen and FFPE tissue blocks. We conclude that although it is indeed true that sequencing data from FFPE tissue can be poorer than those from frozen tissue, any differences occur at an inconsequential magnitude, and FFPE biospecimens can be used in genomic medicine with confidence.

The threat of seed-transmissible pepper yellow leaf curl Indonesia virus in chili pepper.

Recently, chili pepper (Capsicum annuum) plants in Indonesia have been devastated by a notorious bipartite begomovirus infection named Pepper yellow leaf curl Indonesia virus (PepYLCIV), which causes a distinct decrease in chili pepper production. Pepper yellow diseases have been known since early 2000; however, the spread of this virus thus far is distressing. These diseases can reduce chili yields by 20-100% in Indonesia. As previously known, begomovirus can be transmitted through whitefly to several host plants from the families Solanaceae, Compositae, and Leguminosae. In the field, a single plant was observed with severe symptoms of pepper yellow leaf curl disease, while other plants in the same field were asymptomatic and healthy. The observation leads to the possibility that the virus can be transmitted from previously infected chili pepper plants through seeds, as begomovirus transmission through seeds has been reported before. This study was conducted using seeds from chili peppers infected with viruses from different places in Indonesia. Whole seeds, embryos, and seedlings from PepYLCIV infected seeds were investigated in this study by performing viral genome DNA extraction, uracil DNA glycosylase-PCR, and sequencing analysis. Results revealed that both DNA-A and DNA-B of PepYLCIV in seeds and embryos of infected chili pepper plants were detected. The results also showed that 25-67% of PepYLCIV DNA-A and 50-100% of DNA-B were detected from seedlings grown from infected chili pepper seed collected from different location, thus confirming PepYLCIV as a seed-transmissible virus in chili pepper plants.

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

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

High UDG and BRCA1 expression is associated with adverse outcome in patients with pemetrexed treated non-small cell lung Cancer.

OBJECTIVE: The antifolate chemotherapy agent pemetrexed has been widely used to treat non-small-cell-lung-cancer (NSCLC), but there is no clinically validated biomarker to select patients likely to respond. The aim of this study was to assess two proteins involved in DNA repair mechanisms, uracil DNA glycosylase (UDG) and BRCA1 as potential prognostic biomarkers in NSCLC patients treated with pemetrexed-based chemotherapy. MATERIAL AND METHODS: Formalin-fixed-paraffin-embedded tumor specimens from 119 patients with advanced NSCLC treated with pemetrexed between 2004 and 2011 were retrospectively analyzed. Expression of UDG, BRCA1, and known prognostic factors ALK, TTF-1, thymidylate synthase and folylpolyglutamate synthase was assessed by immunohistochemistry using H-SCORE (product of percent stained cells and intensity of expression). Progression-free (PFS) and overall survival (OS) served as reference endpoint. RESULTS: Most NSCLC tumor samples had UDG positivity in at least 5% of tumor cells and 34% samples had more than 50% positive tumor cells. Using the median expression value as threshold, high UDG expression (H-SCORE≥75) was significantly associated with shorter median PFS (3-year PFS 7% vs. 37%, p = 0.045) and a trend for shorter OS (3-year OS 15% vs 42%, p = 0.066) compared to patients with low UDG. In multivariable Cox analysis, the association between high UDG and shorter PFS was close to statistically significant (p = 0.08) at a significance level of 0.05 after controlling for age, gender, ALK- and TTF1-status with hazard ratio of 2.1. Grouping patients according to combined UDG and BRCA1 expression, patients with a profile of UDGhigh/BRCA1high had the shortest PFS and OS compared to all other patient groups (p = 0.007 and 0.02, respectively). CONCLUSION: Our results demonstrate an important prognostic role for high UDG expression in pemetrexed-treated NSCLC patients, in addition to its previously reported role in pemetrexed cytotoxicity. High UDG expression was predictive of shorter PFS and OS, and patients with a combined profile of UDGhigh/BRCA1high had the poorest outcome following pemetrexed treatment.

Awareness of mutational artefacts in suboptimal DNA samples: possible risk for therapeutic choices.

BACKGROUND: Technical biases due to PCR artefacts could represent an insidious obstacle for mutational analysis and precision medicine. METHODS: The authors report a retrospective analysis by fast COLD-PCR and sequencing of 31 suboptimal tumor DNA samples obtained from FFPE tissues and liquid biopsies. RESULTS: In FFPE tumor tissues and plasma liquid biopsies of patients with lung and colorectal adenocarcinoma, we observed a significant rate of artefactual KRAS mutations, unveiled by repeated analysis following UDG pretreatment as well as by simple repetition without UDG pretreatment step, thus suggesting a DNA damage different from cytosine deamination. UDG pretreatment was not only unnecessary to contrast artefacts occurrence, but also hampered the efficiency of mutational screening, reducing the analytical sensitivity. Taken individually or considered together, the reduced DNA input per reaction and UDG pretreatment limited the detection of 'real' mutated alleles, decreasing PCR sensitivity enough to hamper distinction between artefactual and true subclonal mutations of KRAS. CONCLUSIONS: Careful validation of analytical sensitivities should always be carried out through standard controls, and strategies other than UDG pretreatment need to be identified to avoid both amplification of artefactual mutations and failure to identify real subclonal mutations.