Novel target capture DNA library preparation method using CircLigase-mediated hook ligation.

Target capture DNA library preparation methods primarily use the biotin-streptavidin interaction to enrich target DNA, requiring complex operations and a time-consuming workflow with a series of wash steps at regulated temperatures. Here, a new method is presented termed 'hook ligation' for target DNA library preparation, using CircLigase to capture target DNA. The concept was validated by library preparation, sequencing, and acquisition of target DNA fragment information through bioinformatics analysis. An efficient reference point for single-strand DNA ligation to a hook sequence using CircLigase is also provided and it is shown that the efficiency can be influenced by the length and the position of the complementary area on the target DNA.

A novel fluorescence method for activity assay and drug screening of T4 PNK by coupling rGO with ligase reaction.

T4 polynucleotide kinase (PNK) is the primary member of the 5'-kinase family that can transfer the γ-phosphate residue of ATP to the 5'-hydroxyl group of oligonucleotides. In this article, using the differential quenching ability of reduced graphene oxide (rGO) towards the fluorophore-labeled DNA probe, we propose a novel method for detecting T4 PNK activity assisted by ligase reaction. Under the optimized conditions, the detection limit of T4 PNK was estimated to be 0.0002 U µL-1 in the linear region of 0.001 U µL-1-0.1 U µL-1. Additionally, the developed method was used to screen regulators of T4 PNK from natural compounds. The compound f isolated from the root of Kadsura coccinea (Lem.) A.C. Smith was found to stimulate T4 PNK activity in a concentration-dependent manner in vitro. Finally, the method was used to monitor the relation of T4 PNK activity with pelvic inflammatory disease (PID). The results demonstrated that the development of this disease could inhibit T4 PNK activity to some extent. In summary, the above data indicate that the method not only provides a universal platform for monitoring T4 PNK activity, but also shows great potential to be used in drug screening and clinic diagnosis.

Quick Click: The DNA-Templated Ligation of 3'-O-Propargyl- and 5'-Azide-Modified Strands Is as Rapid as and More Selective than Ligase.

The copper(I)-mediated azide-alkyne cycloaddition (CuAAC) of 3'-propargyl ether and 5'-azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate-sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA-templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a kobs value of 1.1 min-1 , comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3'-propargyl ether and 5'-azide-terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.

Comparative analysis of the end-joining activity of several DNA ligases.

DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1) DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase) were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs). This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain) were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.

Targeting the ß-clamp in Helicobacter pylori with FDA-approved drugs reveals micromolar inhibition by diflunisal.

The ß-clamp is the processivity-promoting factor for most of the enzymes in prokaryotic DNA replication; hence, it is a crucial drug target. In the present study, we investigated the ß-clamp from Helicobacter pylori, aiming to seek potential drug molecules against this gastric-cancer-causing bacterium. An in silico screening of Food and Drug Administration (FDA) approved drugs against the H. pylori ß-clamp, followed by its in vitro inhibition using a surface competition approach, yielded the drug diflunisal as a positive initial hit. Diflunisal inhibits the growth of H. pylori in the micromolar range. We determined the structure of diflunisal in complex with the ß-clamp to show that the drug binds at subsite I, which is a protein-protein interaction site. Successful identification of FDA-approved molecules against H. pylori may lead to better and faster drug development.

Discovery and Optimization of NAD+-Dependent DNA Ligase Inhibitors as Novel Antibacterial Compounds.

With the serious problem of multiple drug resistance to antibiotics among pathogenic bacteria spreading across the world over the past 30 years, it is crucial to search for novel inhibitors with distinct modes of action from diverse chemical classes. NAD+-dependent DNA ligases (LigAs) are essential enzymes in bacteria, vital for DNA replication and repair. Additionally, LigAs exclusively exist in eubacteria and some viruses and are not found in humans. Those enzymes have therefore been identified as attractive antibacterial drug targets. In this review we explore the discovered inhibitors of LigA through high-throughput screening or virtual screening respectively and their further structure optimization.

Dietary Calorie Restriction from Adulthood Through Old Age in Rats: Improved DNA Polymerase ß and DNA Gap Repair Activity in Cortical Neurons.

It is well established now that dietary calorie restriction (CR) leads to extension of life span in many species, although the exact mechanism of this effect is still eluding. In the present study, we examined the effect of 40 % CR imposed during a prolonged period of life span (from 6 to 30 months) of rats on the activity of DNA polymerase ß (pol ß) in view of its role in short gap base excision DNA repair and template driven primer extension. DNA pol ß activity is very low at this late age. However, cortical neuronal extracts prepared from CR rats of 30 months age showed significantly higher pol ß protein levels and activity when compared to control 30 month old rats. Yet, one-nucleotide gap repair in old control neurons and an improved efficiency in CR neurons could be visualized only after supplementation of the extracts with T4 DNA ligase indicating the lack of CR affect on ligase activity. No impressive primer extension activity is seen either in the CR or old control neurons. These results are taken to convey that extended CR through adult life leads to improved pol ß activity and therefore, pol ß dependent DNA gap repair activity.

Novel method for screening of radioprotective agents providing protection to DNA ligase against gamma radiation induced damage.

PURPOSE: A simple, sensitive and novel method was developed to screen out potential agents able to protect functional activity of DNA ligase against gamma irradiation-induced damage. Repeatability, authenticity and sensitivity of the method was verified by analyzing DNA ligase protecting activities of well-known radioprotectors such as amifostine, trolox, melatonin, semiquinone glucoside derivative (SQGD) and an antioxidant gallic acid in extremely low concentration (1 µg/reaction). MATERIAL AND METHODS: Two different sets (Set A and B) of T4 DNA ligase (1 unit/set) were prepared. Set 'A' (negative control) was exposed to different doses (3-5 kGy) of gamma radiation in the absence of radioprotective compounds. Set B (test) was exposed to similar doses of gamma radiation in the presence of radioprotective compounds. Following irradiation, DNA ligase was mixed with λ DNA (250 ng) pre-digested with Hind III restriction endonuclease. Ligation reaction was performed in both sets simultaneously at 22°C for 20 min and reaction product was analyzed using agarose gel electrophoresis. RESULTS: Complete DNA ligation was observed in samples where DNA ligase was irradiated in the presence of radioprotectective compounds, i.e., amifostine, trolox, melatonin and a natural radioprotector semiquinone glucoside derivative (SQGD) individually, while, functional impairment in ligation activity of DNA ligase was evident in samples in which DNA ligase was irradiated in the absence of a radioprotective compound. CONCLUSION: The current method was able to provide significant input to screen out radioprotective compounds able to protect DNA ligase functional activity against gamma radiation-induced functional impairment.

Determination of copy number of short tandem repeat using NAD-dependent ligase and pyrosequencing-compatible method.

A sensitive and pyrosequencing-compatible method for determining the copy number of the short tandem repeat (STR) is presented in this study. When Escherichia coli ligase catalyzes the ligation of primer and probes complementary to the proper sites of the target DNA template, it converts nicotinamide adenine dinucleotide to adenosine monophosphate (AMP) and nicotinamide. The AMP release level is proportional to the copy number of the STR and can be measured using adenylate kinase, pyruvate kinase, and luciferase. Unlike current standard methods based on electrophoresis, the present assay is sensitive to the point mutation. Furthermore, after determination of the copy number of the tandem repeat using the proposed method, the DNA templates, primer and probes immobilized onto super paramagnetic beads can be washed and pyrosequencing can be applied for the remaining DNA sequencing. This assay is specially efficient to handle a large number of samples because massively parallel tests could be executed in a microplate photometer. Furthermore, it can work with the pyrosequencing for further sequencing like genome sequencing.

One-step highly sensitive florescence detection of T4 polynucleotide kinase activity and biological small molecules by ligation-nicking coupled reaction-mediated signal amplification.

DNA phosphorylation, catalyzed by polynucleotide kinase (PNK), plays significant regulatory roles in many biological events. Herein, using T4 PNK as a model target, we describe a one-step, highly sensitive, simple and rapid fluorescence approach for monitoring its activity and inhibition. This innovative strategy is inspired by the great amplification capability of ligation-nicking coupled reaction-mediated signal amplification. In the presence of T4 PNK, one of two short oligonucleotides complementary to the loop sequence of molecular beacon (MB) are phosphorylated, and then ligated with the other by DNA ligase. Upon formation of the stable duplex between the ligated DNA and MB, the fluorescence is restored and further significantly amplified through nicking endonuclease assisted cleavage of multiple MBs. Meanwhile, the cleavage of MBs will also generate new nicks to initiate the ligation reaction. Eventually, a maximum fluorescence enhancement is obtained when the ligation and nicking process reached a dynamic equilibrium. As compared to those of the existing approaches except for the assay based on single nanoparticle counting, all limited to 1:1 signal transduction function, the sensitivity (0.00001U/mL) of the proposed strategy is 100-1700 times higher. The application of the sensing system in complex biological matrix and screening of T4 PNK inhibition are demonstrated with satisfactory results. Moreover, this approach is also successfully used to detect biological small molecules such as adenosine triphosphate (ATP), and can be further extended for nicotinamide adenine dinucleotide (NAD(+)) detection.

Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series.

Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins.

While mammalian DNA polymerase ß (Pol ß), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol ß from jellyfish Aurelia sp.1. (AsPol ß). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol ß were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol ß and the other members of the Pol X family.

Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity.

Optimization of adenosine analog inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3'- and 5'-substituents on the ribose. Compounds with logD values 1.5-2.5 maximized potency and maintained drug-like physical properties.

Uracil-DNA glycosylase of Thermoplasma acidophilum directs long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and ATP/ADP, into short-patch repair.

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ∼15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.

Development of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for diagnostic platform.

A multiplex reversal transcription-ligase detection reaction-polymerase chain reaction (MRLP) based universal microarray for the simultaneous pathogens detection was established by using potato viruses as a model. The proposed procedure integrated LDR for multiplicity and specificity, PCR amplification by universal primers for sensitivity, which required design of upstream and downstream LDR probes specific for each virus, and subsequent Zip-code microarray for multiplex and specific identification. Each MRLP fragments carried a unique sequence (complementary Zip-code sequence, cZip-code) which identified a virus by addressed to the location on the microarray where the Zip-code sequence has been spotted. Such Zip-code microarray and universal primers are therefore "universal" being unrelated to a specific molecular analyte. With this technique, target RNAs of ten potato viruses were reversal transcribed by random primer in a single reaction, then subjected to LDR and asymmetric labeling PCR as template, finally, the MRLP amplicons were analyzed by microarray hybridization and subsequent scanning. The technique platform was optimized and evaluated by using reference samples and artificial samples, which can specifically detect down to 3 copies of single or mixed plasmid templates. Due to its universality, multiplexing, specificity and sensitivity, this method can be recommended for simultaneously detecting a large number of different target types in related fields.

Stimulation of DNA repair in Saccharomyces cerevisiae by Ginkgo biloba leaf extract.

Many extracts prepared from plants traditionally used for medicinal applications contain a variety of phytochemicals with antioxidant and antigenotoxic activity. In this work we measured the DNA protective effect of extracts of Ginkgo biloba leaves from oxidative stress using Saccharomyces cerevisiae as experimental model. The extract improved viability of yeast cells under oxidative stress imposed by hydrogen peroxide. In accordance with previous reports on antioxidant properties of G. biloba extracts, pre-incubation of yeast cells promoted a decrease in intracellular oxidation. We assessed DNA damage by our recently developed yeast comet assay protocol. Upon oxidative shock, DNA damage decreased in a dose-dependent manner in experiments of pre-incubation and simultaneous incubation with the extract, indicating a direct protective effect. In addition, the extract improved DNA repair rate following oxidative shock as measured by faster disappearance of comet tails. This suggests that the extract stimulates the DNA repair machinery in its DNA protective action in addition to directly protect DNA from oxidation. The observed DNA repair depends on the DNA repair machinery since no DNA repair was observed under restrictive conditions in a conditional mutant of the CDC9 gene (Accession No. Z74212), encoding the DNA ligase involved in the final step of both nucleotide and base excision repair.

Curcumin prevents DNA damage and enhances the repair potential in a chronically arsenic-exposed human population in West Bengal, India.

Induction of oxidative stress and inhibition of DNA repair are possible modes of arsenic-induced carcinogenesis. In West Bengal, India, several districts contain high levels of arsenic, which are far above the WHO-recommended standard. Prevention of arsenic-induced oxidative stress and induction of repair enzymes by curcumin, an active ingredient of turmeric, may be an effective strategy to combat the adverse effects of arsenic. This study aimed at observing the role of curcumin in reducing 8-hydroxy-20-deoxyguanosine formation and enhancing DNA repair capacity in the arsenic-exposed population of West Bengal. Chronically arsenic-exposed volunteers (n= 66), who were asymptomatic, were selected for this study. Our results indicated that curcumin suppressed the 8-hydroxy-20-deoxyguanosine level and OGG1 expression, which were increased by arsenic. Curcumin also induced DNA repair enzymes involved in both base excision repair and nonhomologous end-joining pathways. In this study, both the protein expression and genetic profile were observed for poly-ADP-ribose polymerase 1, DNA b polymerase, X ray repair cross complement 1, DNA ligase III, DNA protein kinase catalytic sub-unit, X ray repair cross-complement 4, DNA ligase IV, and topoisomerase II b. The results indicated that arsenic-inhibited DNA repair was induced by curcumin, both at protein and genetic levels. Thus, curcumin intervention may be a useful modality for the prevention of arsenic-induced carcinogenesis.

Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase.

An electrochemical method for nicotinamide adenine dinucleotide (NAD(+)) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5'-SH and 3'-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD(+), E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD(+)-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD(+) range from 3 nM to 5 µM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD(+) from its analogues.

Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo.

DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.