Hydroxyl Termini Research Articles

Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies.

Ribozymes are small catalytic RNA sequences capable of nucleotide-specific self-cleavage found widespread in nature. Ribozyme cleavage generates distinct 2',3'-phosphate and 5'-hydroxyl termini that resemble substrates for recently characterized RNA repair pathways in cells. We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for Stitch RNA). Optimization of StitchR activity in mammalian cells resulted in a ~900-fold increase in protein expression that approached levels observed for genes expressed from single vectors. We demonstrate that StitchR can be harnessed for effective dual adeno-associated virus gene therapies to correct muscular dystrophies by restoring large functional muscle proteins to endogenous levels in vivo.

Construction of an efficient microRNA sensing platform based on terminal deoxynucleotidyl transferase-mediated synthesis of copper nanoclusters

A conceptual innovative electrochemical sensing platform was constructed for microRNA (miRNA) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated synthesis of copper nanoclusters. In principle, the substrate strand immobilized on working electrode acted as the template for the TdT-mediated DNA extension reaction, while the DNAzyme probe would be activated in the absence of miRNA 21, which led to the cleavage of the substrate strand to produce the 5’ phosphate terminus and inhibited the TdT-mediated DNA extension reaction. On the other hand, the presence of miRNA 21 initiated the toehold-mediated strand displacement reaction, causing the disassembly of the DNAzyme structure and preventing the substrate strand from being cleaved. This enabled the recycle amplification of target miRNA 21, and TdT catalyzed the DNA extension reaction on the 3’ hydroxyl terminus of the substrate strand, producing a large number of poly thymine (polyT) sequences. These sequences bound with copper ions for in-situ synthesis of copper nanoclusters on the sensing electrode, and the increased electrochemical signal of copper was recorded by differential pulse stripping voltammetry (DPSV), which paved the way for sensitive determination of miRNA 21 extracted from cancer cells with a detection limit of 36 aM. Due to its high sensitivity, user-friendly operation, and cost-effectiveness, this method is helpful for the fundamental research of sensing mechanism as well as the diagnosis of related diseases.

Pluronic-Based Nanoparticles for Delivery of Doxorubicin to the Tumor Microenvironment by Binding to Macrophages.

The active targeting drug delivery system based on special types of endogenous cells such as macrophages has emerged as a promising strategy for tumor therapy, owing to its tumor homing property and biocompatibility. In this work, the active tumor-targeting drug delivery system carrying doxorubicin-loaded nanoparticles (DOX@MPF127-MCP-1, DMPM) on macrophage (RAW264.7) surfaces via the mediation of interaction with the CCR2/MCP-1 axis was exploited. Initially, the amphiphilic block copolymer Pluronic F127 (PF127) was carboxylated to MPF127 at the hydroxyl terminus. Subsequently, MPF127 was modified with MCP-1 peptide to prepare MPF127-MCP-1 (MPM). The DOX was wrapped in MPM to form DMPM nanomicelles (approximately 100 nm) during the self-assembly process of MPM. The DMPM spontaneously bound to macrophages (RAW264.7), which resulted in the construction of an actively targeting delivery system (macrophage-DMPM, MA-DMPM) in vitro and in vivo. The DOX in MA-DMPM was released in the acidic tumor microenvironment (TME) in a pH-responsive manner to increase DOX accumulation and enhance the tumor treatment effect. The ratio of MA-DMPM homing reached 220% in vitro compared with the control group, indicating that the MA-DMPM was excellently capable of tumor-targeting delivery. In in vivo experiments, nonsmall cell lung cancer cell (NCI-H1299) tumor models were established. The results of the fluorescence imaging system (IVIS) showed that MA-DMPM demonstrated tremendous tumor-targeting ability in vivo. The antitumor effects of MA-DMPM in vivo indicated that the proportion of tumor cell apoptosis in the DMPM-treated group was 63.33%. The findings of the tumor-bearing mouse experiment proved that MA-DMPM significantly suppressed tumor cell growth, which confirmed its immense potential and promising applications in tumor therapy.

Roles of O-GlcNAcylation in Mitochondrial Homeostasis and Cardiovascular Diseases.

Protein posttranslational modifications are important factors that mediate the fine regulation of signaling molecules. O-linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is a monosaccharide modification on N-acetylglucosamine linked to the hydroxyl terminus of serine and threonine of proteins. O-GlcNAcylation is responsive to cellular stress as a reversible and posttranslational modification of nuclear, mitochondrial and cytoplasmic proteins. Mitochondrial proteins are the main targets of O-GlcNAcylation and O-GlcNAcylation is a key regulator of mitochondrial homeostasis by directly regulating the mitochondrial proteome or protein activity and function. Disruption of O-GlcNAcylation is closely related to mitochondrial dysfunction. More importantly, the O-GlcNAcylation of cardiac proteins has been proven to be protective or harmful to cardiac function. Mitochondrial homeostasis is crucial for cardiac contractile function and myocardial cell metabolism, and the imbalance of mitochondrial homeostasis plays a crucial role in the pathogenesis of cardiovascular diseases (CVDs). In this review, we will focus on the interactions between protein O-GlcNAcylation and mitochondrial homeostasis and provide insights on the role of mitochondrial protein O-GlcNAcylation in CVDs.

Extraordinarily Stable Hairpin-Based Biosensors for Rapid Detection of DNA Ligases.

DNA ligases are essential enzymes involved in DNA replication and repair processes in all organisms. These enzymes seal DNA breaks by catalyzing the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in double-stranded DNA. In addition to their critical roles in maintaining genomic integrity, DNA ligases have been recently identified as diagnostic biomarkers for several types of cancers and recognized as potential drug targets for the treatment of various diseases. Although DNA ligases are significant in basic research and medical applications, developing strategies for efficiently detecting and precisely quantifying these crucial enzymes is still challenging. Here, we report our design and fabrication of a highly sensitive and specific biosensor in which a stable DNA hairpin is utilized to stimulate the generation of fluorescence signals. This probe is verified to be stable under a wide range of experimental conditions and exhibits promising performance in detecting DNA ligases. We anticipate that this hairpin-based biosensor will significantly benefit the development of new targeting strategies and diagnostic tools for certain diseases.

Intramolecular Accelerated Assembly of Molecular Beacons: A DNA Nanoarchitecture-based Spatial Confinement Strategy toward Terminal Deoxynucleotidyl Transferase Biosensing.

Physiological function analysis of terminal deoxynucleotidyl transferase (TdT) in clinical medicine and hematopathology highlights its significance to be extensively utilized as a diagnostic biomarker for leukemia diagnosis. Herein, taking advantage of the spatial-confinement effect on a three-dimensional (3D) DNA nanoarchitecture, we reported a target-triggered intramolecular accelerated molecular beacon (MB) assembly for rapid and real-time analysis of TdT activity. In this strategy, the 3D DNA nanoarchitecture is first engineered via a cross-linking network hybridization chain reaction (HCR). A number of MBs, which were designed with a polythymine (poly-T) loop, were then conjugated on the scaffold DNA nanoarchitecture, allowing the obtained MB-DNA nanoarchitecture to contain lots of free 3'-hydroxyl (OH) termini inside or outside the super DNA nanostructure. Moreover, the distance between different MBs is closed, and the local concentration of MB is significantly improved owing to the confinement of MBs on this DNA nanoarchitecture. Once encountered with target TdT, the free -OH groups can be recognized by TdT immediately to catalyze the template-independent incorporation of adenine nucleotides, which results in the generation of multiple poly-A chains that rapidly react with many MBs via an intramolecular accelerated assembly process. The time-dependent substantial enhancement of the fluorescence from MBs can thus be applied for robustly analyzing TdT. Our observations suggest that the DNA nanostructure-based spatial confinement effect enables a high molecular collision frequency to accelerate the reaction kinetics, and the super DNA nanoarchitecture exhibits a better nuclease resistance to maintain signal stability. With these advantages, TdT can be rapidly detected with high sensitivity, specificity, and biostability.

TtAgo sensor for the sensitive and rapid detection of T4 polynucleotide kinase activity

As a next-generation biosensing technology, the application of Argonaute for analytical science research has attracted considerable attention. A Thermus thermophilus Argonaute (TtAgo)-based sensor is presented for detecting T4 polynucleotide kinase (PNK) activity considering that TtAgo’s multiple-turnover catalytic activity can be activated by binding to a 5′-phosphate termini of single-stranded DNA (ssDNA) for the cleavage of complementary strand. PNK catalyzes the phosphorylation of 5′-hydroxyl termini of ssDNA, and the phosphorylation product termed guide can activate the TtAgo-mediated complementary ssDNA reporter cleavage to release fluorescence signal. For the PNK activity monitoring, the developed method shows good analytic performance with a linear range of 0.0010–2 U mL−1 and a detection limit of 0.0010 U mL−1 without the use of a preamplification reaction. This method is successfully applied for the analysis of HeLa cells at a single-cell level. The developed method shows several advantages, including high sensitivity, low cost, facile operation, and has remarkable potential application for early disease diagnosis, drug screening, and clinical treatment.

Fluorescence in situ Localization of Pri-miRNAs in Isolated Arabidopsis thaliana Nuclei.

Here, we present an approach combining fluorescence in situ hybridization (FISH) and immunolabeling for localization of pri-miRNAs in isolated nuclei of A. thaliana. The presented method utilizes specific DNA oligonucleotide probes, modified by addition of digoxigenin-labeled deoxynucleotides to its 3' hydroxyl terminus by terminal deoxynucleotidyl transferase (TdT). The probes are then detected by immunolabeling of digoxigenin (DIG) using specific fluorescent-labeled antibodies to visualize hybridized probes. Recently, we have applied this method to localize pri-miRNA156a, pri-miRNA163, pri-miRNA393a, and pri-miRNA414 in the nuclei isolated from leaves of 4-week-old A. thaliana. The present approach can be easily implemented to analyze nuclear distribution of diverse RNA classes, including mRNAs and pri-miRNAs in isolated fixed cells or nuclei from plant.

Programmable CRISPR-Cas12a and self-recruiting crRNA assisted dual biosensing platform for simultaneous detection of lung cancer biomarkers hOGG1 and FEN1

Human 8-oxoguanine DNA glycosylase (hOGG1) and flap endonuclease 1 (FEN1) are recognized as potential biomarkers in lung cancer investigations. Developing analytical platforms of simultaneously targeting hOGG1 and FEN1 with high selectivity, sensitivity, especially programmability and universality is highly valuable for clinical research. Herein, we established a signal-amplified platform for simultaneously detecting hOGG1 and FEN1 on the basis of cleavage-induced ligation of DNA dumbbell probes, rolling circle transcription (RCT) and CRISPR-Cas12a. A hOGG1 cleavable site and FEN1 cleavable flap were dexterously designed at the 5’ end of DNA flapped dumbbell probes (FDP) for hOGG1 and FEN1. After cleavage, the resulting nick sites with juxtaposition of 5′ phosphate and 3′ hydroxyl terminus could be linked to closed DNA dumbbell probes (CDP) by DNA ligase. The CDP served as a template for RCT, producing plentiful crRNA repeats to activate the trans-cleavage activity of CRISPR-Cas12a which could cleave fluorophores (TAMRA and FAM) and quenchers (BHQ2 and BHQ1) double-labeled ssDNA reporters. Then, hOGG1 and FEN1 could be detected by the recovered fluorescence signal, allowing for the highly sensitive calculated detection limits of 0.0013 and 0.0052 U/mL, respectively. Additionally, this method made it possible to evaluate the inhibitory effects, even to measure hOGG1 and FEN1 activities at the single-cell level. This novel target enzyme-initiated, circles-transcription without promoters, real-time generation, and self-assembly features of FDP-RCT-Cas12a system suppressed nonspecific background remarkably and relieved rigorous requirement of protospacer adjacent motif site. Hence, the universality of FDP-RCT-Cas12a system toward various disease-related non-nucleic acid targets which are tested without using aptamers was extremely improved.

A label-free fluorescent biosensor for amplified detection of T4 polynucleotide kinase activity based on rolling circle amplification and catalytic hairpin assembly

T4 polynucleotide kinase (PNK) plays a key role in maintaining genome integrity and repairing DNA damage. In this paper, we proposed a label-free fluorescent biosensor for amplified detection of T4 PNK activity based on rolling circle amplification (RCA) and catalytic hairpin assembly (CHA). Firstly, we designed a padlock probe with a 5′-hydroxyl terminus for phosphorylation reaction, a complementary sequence of the primer for initiating RCA, and a complementary sequence of the trigger for triggering CHA. T4 PNK catalyzed the phosphorylation reaction by adding a phosphate group to the 5′-hydroxyl terminus of padlock probe, generating a phosphorylated padlock probe. Then it hybridized with the primer to generate a circular probe under the action of ligase. Subsequently, the primer initiated an RCA reaction along the circular probe to synthesize a large molecular weight product with repetitive trigger sequences. The triggers then triggered the cyclic assembly reactions between hairpin probe 1 and hairpin probe 2 to generate a large amount of complexes with free G-rich sequences. The free G-rich sequences folded into G-quadruplex structures, and the N-methylmesoporphyrin IXs were inserted into them to produce an amplified fluorescent signal. Benefiting from high amplification efficiency of RCA and CHA, this fluorescent biosensor could detect T4 PNK as low as 6.63 × 10−4 U mL−1, and was successfully applied to detect its activity in HeLa cell lysates. Moreover, this fluorescent biosensor could effectively distinguish T4 PNK from other alternatives and evaluate the inhibitory effect of inhibitor, indicating that it had great potential in drug screening and disease treatment.

Microbial Surfactants: Bright Prospects for Innovation, New Product Categories, and Commercialization

Microbial Surfactants: Bright Prospects for Innovation, New Product Categories, and Commercialization

A multiple primers-mediated exponential rolling circle amplification strategy for highly sensitive detection of T4 polynucleotide kinase and T4 DNA ligase activity

T4 polynucleotide kinase (T4 PNK) and T4 DNA ligase are two important repair enzymes that play a significant role in maintaining the stability and integrity of genome. Here, we proposed a multiple primers-mediated exponential rolling circle amplification strategy for label-free and highly sensitive fluorescent analysis of T4 PNK and T4 DNA ligase activity. Firstly, we designed a target strand that contained two C-rich sequences, a complementary sequence of the primer, and three cleavage sites of Nt.BbvCI scattered among the three sequences. The 5′-hydroxyl terminus of target strand was catalyzed by T4 PNK to form a phosphorylated target strand. After the phosphorylated target strand was combined with the primer, T4 DNA ligase catalyzed the formation of circular DNA. Then, the primer triggered an exponential rolling circle amplification reaction along the circular DNA template, exponentially generating G-rich sequences. N-methyl mesoporphyrin IX as signal molecules specifically bound to the G-quadruplexs folded by G-rich sequences, generating a detectable fluorescent signal. Benefiting from exponential signal growth, this strategy showed extremely high detection sensitivity with detection limits of 8.11 × 10−5 U mL−1 of T4 PNK and 3.07 × 10−4 U mL−1 of T4 DNA ligase, respectively. In addition, the proposed strategy exhibited excellent inhibitor evaluation effects for two repair enzymes, and could detect T4 PNK in HeLa cell lysate. The obtained results showed that the proposed strategy had excellent application prospects in disease diagnosis and treatment evaluation.

General Approach to N6,C5'-Difunctionalization of Adenosine.

Among the C6-halo purine ribonucleosides, the readily accessible 6-chloro derivative has been known to undergo slow SNAr reactions with amines, particularly aryl amines. In this work, we show that in 0.1 M AcOH in EtOH, aryl amines react quite efficiently at the C6-position of 2',3',5'-tri-O-(t-BuMe2Si)-protected 6-chloropurine riboside (6-ClP-riboside), with concomitant cleavage of the 5'-silyl group. These two-step processes proceeded in generally good yields, and notably, reactions in the absence of AcOH were much slower and/or lower yielding. Corresponding reactions of 2',3',5'-tri-O-(t-BuMe2Si)-protected 6-ClP-riboside with alkyl amines proceeded well but without desilylation at the primary hydroxyl terminus. These differences are likely due to the acidities of the ammonium chlorides formed in these reactions, and the role of AcOH was not desilylation but possibly only purine activation. With 50% aqueous TFA in THF at 0 °C, cleavage of the 5'-silyl group from 2',3',5'-tri-O-(t-BuMe2Si)-protected N6-alkyl adenosine derivatives and from 6-ClP-riboside was readily achieved. Reactions of the 5'-deprotected 6-ClP-riboside with alkyl amines proceeded in high yields and under mild conditions. Because these complementary methodologies yielded N6-aryl and -alkyl adenosine derivatives containing a free 5'-hydroxyl group, a variety of product functionalizations were undertaken to yield N6,C5'-doubly modified nucleoside analogues.

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5'-Terminal Structure.

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

Lignite powder briquetting with binder and adsorption model of binder on the coal surface

ABSTRACT The addition of different binders can improve the mechanical strength of briquette for the lignite powder. The changes of lignite in wettability, functional groups and surface electrical properties were analyzed after briquetting. The model of the interaction between the binder and coal particle was studied. The results revealed that starch and polyvinyl alcohol binders could make the compressive and drop strength of lignite briquette strong. When the potential value decreased, it was beneficial to the briquetting process. The Fourier transform infrared spectra (FTIR) analysis showed that functional groups of binder adsorbed the oxygen-containing functional groups of coal. After the binder interacted with the pulverized coal, the molecules in the binder were adsorbed on the surface of coal, which changed the hydrophobicity of the pulverized coal. An adsorption model was proposed as follows: The first was the various types of hydrogen bonds between the hydroxyl terminus of binder and the carboxyl group or hydroxyl group on the surface of coal. The second was the generation of hydrogen bonds among the binder molecules. The greater hydrogen bonding was, the higher the strength of briquette was.

Primer-template conversion-based cascade signal amplification strategy for sensitive and accurate detection of polynucleotide kinase activity

Here, a primer-template conversion-based cascade signal amplification strategy is described for the sensitive detection of polynucleotide kinase (PNK) activity. This strategy integrated rolling circle amplification (RCA) and multiple-repeated-strand displacement amplification (MRSDA) with G-quadruplex based fluorescence lighting-up assay. A delicate dumbbell-shaped DNA probe with 5′-hydroxyl terminus was designed, in which G-quadruplex and half recognition site of nicking enzyme Nb.BbvCI were encoded in two loops respectively. Under the action of PNK, the 5′ terminus on dumbbell probe was firstly phosphorylated, and then the dumbbell was cyclized with the catalyzation of T4 ligase to become the RCA template. The RCA process produced multiple copies of the prolonged primer. After that, under the assistance of nicking enzyme Nb.BbvCI, a primer-template conversion occurred, which converted the primer and template of RCA into the template and primer of the subsequent MRSDA, respectively. The MRSDA generated multiple repeated ssDNA sequences which possessed G-quadruplexes for outputting signal by lighting-up fluorescence of thioflavin T (ThT). The cascade signal amplification of RCA and MRSDA provided high detection sensitivity, and the target-dependence of template in cascade signal amplification led to a low background. The method showed excellent detection limit of 0.2 × 10−6 U μL−1 in buffer and 5 cells in cell lysate sample. Moreover, this method displayed favorable selectivity when interfering proteins were present. The developed strategy has good practical potential for PNK activity detection in clinical diagnosis and medical research.

Labeling the 3' Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT, also simply called terminal transferase) is a template-independent polymerase that catalyzes the addition of deoxynucleotides and dideoxynucleotides to the 3'-hydroxyl terminus of a DNA molecule. Cobalt (Co2+) is a necessary cofactor for the activity of this enzyme. Incorporation at the 3' terminus can be limited to just 1 nt by using [α-32P]ddATP or biotin-, digoxigenin (DIG)-, or fluorescein-ddUTP. Because none of these molecules carries a 3'-hydroxyl group, no additional molecules can be incorporated. Alternatively, the enzyme is capable of adding several (2-100) nt to 3' ends in a so-called homopolymeric "tailing" reaction. A tailing reaction is performed in the presence of a mixture of labeled and unlabeled dNTPs. The rate of addition of dNTPs, and thus the length of the tail, is a function of the ratio of 3' DNA ends to dNTP concentration and, in addition, the specific dNTP that is used.

Discovery of potential inhibitors targeting the kinase domain of polynucleotide kinase/phosphatase (PNKP): Homology modeling, virtual screening based on multiple conformations, and molecular dynamics simulation

In recent years, the level of interest has been increased in developing the DNA-repair inhibitors, to enhance the cytotoxic effects in the treatment of cancers. Polynucleotide kinase/phosphatase (PNKP) is a critical human DNA repair enzyme that repairs DNA strand breaks by catalyzing the restoration of 5’-phosphate and 3’-hydroxyl termini that are required for subsequent processing by DNA ligases and polymerases. PNKP is the only protein that repairs the 3′-hydroxyl group and 5′-phosphate group, which depicts PNKP as a potential therapeutic target. Besides, PNKP is the only DNA-repair enzyme that contains the 5′-kinase activity, therefore, targeting this kinase domain would motivate the development of novel PNKP-specific inhibitors. However, there are neither crystal structures of human PNKP nor the kinase inhibitors reported so far. Thus, in this present study, a sequential molecular docking-based virtual screening with multiple PNKP conformations integrating homology modeling, molecular dynamics simulation, and binding free energy calculation was developed to discover novel PNKP kinase inhibitors, and the top-scored molecule was finally submitted to molecular dynamics simulation to reveal the binding mechanism between the inhibitor and PNKP. Taken together, the current study could provide some guidance for the molecular docking based-virtual screening of novel PNKP kinase inhibitors.

High-Sensitive Detection of Small-Cell Lung Cancer Cells Based on Terminal Deoxynucleotidyl Transferase-Mediated Extension Polymerization Aptamer Probe.

Small-cell lung cancer (SCLC) is characterized by early metastasis and high invasiveness, poor prognosis, and a low five-year survival rate. Therefore, the development of the effective detection of SCLC cells and imaging methods has potential significance for the prognosis and treatment of SCLC. We designed a terminal deoxynucleotidyl transferase (TdT)-mediated extension polymerization aptamer probe (denoted as TEPAP). Aptamer HCC03 was used as an element of recognizing SCLC, and it was extended as a long poly(T) tail at the 3'-hydroxyl terminus by TdT and then hybridized with short poly(A) labeled with 6-carboxyfluorescein (FAM) to construct TEPAP for the high-sensitivity detection of SCLC. The results showed that the probe could specifically recognize NCI-H446 cells. Compared with HCC03 labeled with FAM, TEPAP has demonstrated a higher fluorescence signal in recognizing NCI-H446 cells, and the fluorescence intensity of TEPAP recognizing the target cells was 10 times higher than that of nontarget cells. Flow cytometric analysis showed that the detection limit of this method was as low as 17 NCI-H446 cells in 200 μL of binding buffer. In the application of clinical cytology cell blocks, the sensitivity, specificity, and accuracy of TEPAP were 89.74, 94.44, and 91.23%, respectively. The high sensitivity and specificity of TEPAP in the application of clinical samples show that the proposed probe has great potential in the diagnosis of SCLC.

Ligase 1 is a predictor of platinum resistance and its blockade is synthetically lethal in XRCC1 deficient epithelial ovarian cancers.

Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5′-phosphoryl and 3′-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown.Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells.Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids.Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.